A salt-tolerant glutaminase with improved stability

Abstract

The present invention discloses a salt-tolerant glutaminase with improved stability, belonging to the field of enzyme engineering technology. Through computer-assisted rational design, the present invention screened and obtained a mutant, H105K, in which the histidine 105th residue of the wild-type glutaminase was mutated to lysine. Compared with the wild-type enzyme, the H105K mutant improved its thermal stability by 247.36%, without significantly altering its salt tolerance. This mutant may help expand the application of glutaminase in the food industry.

Description

Technical Field

[0001] The invention relates to salt-resistant glutaminase with improved stability, and belongs to the technical field of enzyme engineering. Background Art

[0002] L-glutaminase (EC 3.5.1.2) catalyzes the hydrolysis of L-glutamine into L-glutamate and ammonia and is widely distributed in microorganisms such as bacteria, yeast, and fungi. This enzyme has great potential for application in food and medicine. The L-glutamate produced by this reaction is the primary substance responsible for the fresh flavor of food. Approximately 46% of the glutamate in brewed soy sauce is produced from L-glutamate during the soy sauce fermentation process. However, the high salt environment in soy sauce inhibits L-glutaminase activity in Aspergillus koji, resulting in the production of more odorless pyroglutamine rather than the flavor-producing glutamate. Therefore, the development of salt-tolerant L-glutaminases is crucial to increasing the L-glutamate content in soy sauce. L-glutaminase from Bacillus sp. LKG-01 (MTCC 10401), isolated from Gangotri district in Uttarakhand in the Himalayas, exhibits relatively stable enzyme activity even at a salt concentration of 25%. L-glutaminase from Micrococcus luteus K-3 exhibits maximum enzymatic activity in the presence of 1.71 M NaCl, while it exhibits over 90% activity in the presence of 3.08 M NaCl. Under optimal conditions, the enzyme activity reaches 1603 U / mg, but after incubation at 50°C for 10 minutes, the activity decreases by 80%, indicating poor temperature stability. Furthermore, the enzyme from this source poses certain food safety risks. Therefore, screening for temperature-stable and food-safe L-glutaminase has important applications in food production. Summary of the Invention

[0003] The present invention provides a glutaminase mutant derived from Lactobacillus reuteri. The glutaminase mutant has at least one of the following mutations (1) to (7) based on the glutaminase shown in SEQ ID NO.1:

[0004] (1) Mutate asparagine at position 2 to tryptophan;

[0005] (2) mutating the threonine at position 21 to aspartic acid;

[0006] (3) mutating aspartic acid at position 44 to glycine;

[0007] (4) mutating glutamic acid at position 59 to glutamine;

[0008] (5) mutating aspartic acid at position 93 to asparagine;

[0009] (6) mutating histidine 105 to lysine;

[0010] (7) mutating serine at position 247 to alanine;

[0011] (8) Mutate aspartic acid at position 302 to asparagine.

[0012] In one embodiment, the glutaminase mutant is based on the glutaminase shown in SEQ ID NO.1, and the histidine at position 105 is mutated to lysine to obtain the glutaminase mutant H105K having the amino acid sequence shown in SEQ ID NO.2.

[0013] In one embodiment, the glutaminase mutant is based on the glutaminase shown in SEQ ID NO. 1, wherein the serine at position 247 is mutated to alanine to obtain the mutant S247A.

[0014] The present invention also provides a gene encoding the glutaminase mutant.

[0015] In one embodiment, the gene encoding the glutaminase mutant has a nucleotide sequence as shown in SEQ ID NO.3.

[0016] The invention also provides a recombinant plasmid carrying the gene.

[0017] In one embodiment, the recombinant plasmid is the plasmid pMA5 to which the gene is connected.

[0018] The present invention also provides a recombinant microbial cell carrying the gene or containing the recombinant plasmid.

[0019] In one embodiment, the recombinant microbial cell uses Bacillus subtilis as a host cell.

[0020] In one embodiment, the host cell includes but is not limited to Bacillus subtilis 168.

[0021] The present invention also provides a method for preparing the glutaminase mutant, which comprises culturing a recombinant microorganism expressing the glutaminase mutant in a culture medium for a period of time and collecting glutaminase in the cell culture fluid.

[0022] In one embodiment, the method is to culture the recombinant Bacillus subtilis expressing the glutaminase mutant in LB medium at 35-39°C and 150-220r / min for 10-12h, transfer it to LB medium, culture it at 28-35°C and 150-220r / min for 20-30h, centrifuge the fermentation broth to collect the bacteria, wash the cells with PBS solution to disrupt the bacteria, and collect the supernatant by centrifugation to obtain the glutaminase mutant.

[0023] The present invention also provides a use of the glutaminase mutant or the recombinant Bacillus subtilis in preparing food.

[0024] Beneficial effects:

[0025] The present invention provides a glutaminase mutant with improved thermal stability, which can increase the enzyme activity of the mutant H105K to 2.47 times that of the wild enzyme after incubation at 37°C for 72 hours, and does not significantly change the salt tolerance, which helps to broaden its application range in the food field. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Enzymatic properties of the wild-type enzyme LreuglsA; (A) Effect of temperature on LreuglsA activity; (B) Effect of pH on LreuglsA activity; (C) Effect of stability at different temperatures on LreuglsA activity; (D) Enzyme kinetics fitting; Michaelis–Menten equation using Origin's nonlinear fitting Hill function (n=1); (E) Effect of metal ions on LreuglsA activity; (F) Effect of salt concentration on LreuglsA glutaminase activity.

[0027] Figure 2 The relative enzyme activity of the mutants after reaction at 55°C for 5 minutes.

[0028] Figure 3 Relative enzyme activities of mutants after incubation at 37°C for 72 h. DETAILED DESCRIPTION

[0029] Bacillus subtilis 168 involved in the following examples was purchased from Sangon Biotech (Shanghai) Co., Ltd.; pMA5 plasmid involved in the following examples was purchased from BioVector China Plasmid Vector Strain Cell Gene Collection Center.

[0030] The culture medium involved in the following examples is as follows:

[0031] (1) LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0032] (2) LB solid medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L.

[0033] (3) TB culture medium:

[0034] A: Yeast powder 24g, peptone 12g, glycerol 4g

[0035] B: KH2PO4 2.3g, K2HPO4 16.4g

[0036] A was dissolved in 900 mL of ultrapure water and sterilized by autoclave; B was dissolved in 100 mL of ultrapure water and sterilized by autoclave, and 900 mL of A and 100 mL of B were mixed to prepare 1 L of TB medium.

[0037] The detection methods involved in the following embodiments are as follows:

[0038] The activity of L-glutaminase is calculated by measuring the L-glutamate produced. The reaction mixture (1 mL) contains 880 μL of 200 mM L-glutamine and 20 μL of L-glutaminase. The reaction is carried out at a specific temperature for 5 minutes and terminated by adding 100 microliters of 15% (w / v) trichloroacetic acid (TCA). After centrifugation and filtration, the L-glutamate in the supernatant is measured using a biosensor analyzer (Institute of Biology, Shandong Academy of Sciences). One unit (U) of L-glutaminase enzyme activity is defined as the amount of enzyme required to produce 1 μmol of L-glutamate per minute.

[0039] Example 1: Preparation of wild-type enzyme and determination of enzymatic properties

[0040] (1) Expression of wild-type glutaminase

[0041] A glutaminase gene lglsA with a nucleotide sequence as shown in SEQ ID NO. 4 was synthesized, the gene lglsA and the pMA5 vector were double-digested with enzymes MluI and NdeI, the digested products were ligated using homologous recombinases, and amplified by Escherichia coli JM109. The recombinant vector pMA5-lglsA was obtained by plasmid extraction; and the recombinant plasmid PMA5-glsA was transformed into Bacillus subtilis 168 to obtain strain BSW1. The recombinant strain BSW1 was inoculated into 10 mL of LB medium and cultured at 37°C and 180 rpm for 10 hours. The strain was then transferred to 50 mL of LB medium at a 1% inoculum and cultured at 30°C and 180 rpm for 30 hours to express glutaminase. The bacterial suspension was harvested, washed with PBS, and collected by centrifugation. 30 μL of lysozyme (200 mg / mL) was added to the cells and sonicated at 400W for 2 seconds, 5 seconds, and then centrifuged at 12,000 rpm for 20 minutes at 4°C. The supernatant and precipitate were separated and filtered through a 0.45 μm filter. A His tag was inserted into the N-terminus of LreuglsA. Expression levels were verified by SDS-PAGE, and the crude enzyme was purified by Ni-NTA affinity chromatography and washed multiple times with MO (containing 20 mM Tris and 500 mM NaCl). The protein was then eluted with increasing concentrations of M500 (containing 20 mM Tris, 500 mM NaCl, and 500 mM imidazole) according to the manufacturer's protocol (GE Healthcare Bio-Sciences). The purified LreuglsA and crude enzyme were analyzed using SDS-PAGE analysis, which showed that the enzyme protein had a molecular weight of approximately 30 kDa.

[0042] (2) Determination of enzymatic properties of glutaminase:

[0043] (a) Enzymatic reactions were performed under different temperature and pH conditions:

[0044] The assay was performed using the SBA biosensor analyzer from the Institute of Biology, Shandong Academy of Sciences. The total volume of the glutaminase activity assay system was 1 mL, containing a 200 mM glutamine solution and 880 μL of buffer at a final concentration. The solution was preheated at different temperatures (20-60°C) for 5 minutes, and 20 μL of crude enzyme solution was quickly added and mixed. The solution was reacted at the corresponding temperature for 5 minutes, and 100 μL of 15% trichloroacetic acid solution was quickly added and mixed to terminate the reaction. For the control, 20 μL of crude enzyme solution was quickly added and mixed, followed by 100 μL of 15% trichloroacetic acid solution and mixed. The buffer was used to control the pH of the reaction system. For pH 4-6, 50 mmol acetate buffer was used, for pH 6-7, 50 mmol phosphate buffer was used, for pH 7-9, 50 mmol Tris-HCl buffer was used, and for pH 9-10, 50 mmol glycine buffer was used.

[0045] At room temperature, 1 mL of the reaction system was centrifuged at 10,000 rpm for 10 minutes to allow the protein precipitate to adhere to the bottom of the centrifuge tube. The glutamate concentration in the supernatant was diluted to 0.3-0.7 g / L. 25 μL of the supernatant was pipetted into the SBA biosensor to measure the glutamate content in the supernatant and calculate the glutaminase activity.

[0046] The results showed that the specific activity of the purified enzyme was 1048.14±7.83 U / mg at 50°C and pH 7.5 (50mM Tris-HCl buffer). In the absence of any protective agent, the enzyme remained stable (activity remained above 60%) for 216 hours at 4°C and pH 6-7. In terms of temperature stability, the enzyme achieved a half-life of approximately 44 hours at the industrial operating temperature of 37°C. 1 / 2 Under the optimal reaction conditions of 50°C and pH 7.5 (50mM Tris-HCl buffer), the enzyme's kinetic constant Km was 51.24±4.39mM and Vmax was 7.84±0.33mM / min. We studied the effect of adding 1mM metal ions on the enzyme activity. + It has an activating effect on enzymes, while other metal ions inhibit the activity of enzymes to a certain extent (less than 30%).

[0047] (b) Enzymatic reactions were performed at different salt concentrations:

[0048] Glutamine enzyme reaction systems containing different concentrations of NaCl were prepared separately. Specifically, in a 1000 μL reaction system, the final concentrations were: NaCl 0-300 g / L (a concentration gradient of 50 g / L), glutamine 200 mM, Tris-HCl buffer 50 mM, and enzyme protein 10 mg / mL. The reaction was carried out at 50°C for 5 min, and the production of glutamate was analyzed. The glutaminase activity measured without the addition of NaCl (NaCl content of 0) was determined to be 100%.

[0049] The results showed that the activity of LreuglsA reached its peak at a salt concentration of 5%, reaching 110.03% of the activity under blank conditions. At a concentration close to saturation (30%) of NaCl, 73.85% of the activity under blank conditions was maintained. Figure 1 These data suggest that LreuglsA has good potential for industrialization. Its ability to maintain the necessary activity under high-salt conditions suggests that it could be used in high-salt industrial brewing processes, such as the soy sauce brewing process.

[0050] Example 2: Design of mutation sites

[0051] PSSMs, representing conserved residues, were generated using psiblast in NCBI-blast-2.9. By analyzing the conservation of these residues and the evolutionary information derived from the PSSMs, the evolvability of the L-glutaminase family and other homologous proteins was investigated for all positions within the protein. Conserved residues were assigned higher scores at the corresponding positions. Based on the PSSM scores, evolvable residues were selected as target sites for mutation. FoldX was used to predict the overall stability of virtual saturation mutations at all sites, and the thermal stability of the protein was predicted by comparing the Gibbs energy changes (ddG, ΔΔG) after mutation. Conserved residues identified in the PSSM analysis were removed from the candidate list. Undesirable interactions within the protein may lead to increased structural instability. Molecular dynamics (MD) simulations describe the dynamic changes of the system at the atomic level by calculating the atomic motions of the protein in solvent, visually demonstrating the mechanisms and principles underlying experimental observations. The root mean square fluctuation (RMSF) value calculated from MD simulations is used to indicate the flexibility of amino acid positions. Finally, eight mutation sites were selected based on computer-assisted mutation prediction combined with visual inspection: position 2 (N2W), position 21 (T21D), position 44 (D44G), position 59 (E59Q), position 93 (D93N), position 105 (H105K), position 247 (S247A), and position 302 (D302N).

[0052] Example 3: Construction of mutant strains

[0053] Primers were designed and pMA5-lglsA was used as a template to amplify the gene sequence with N2W, T21D, D44G, E59Q, D93N, 1H105K, 247S247A and D302N mutations.

[0054] The recombinant plasmid was constructed and expressed in Bacillus subtilis 168 using the same method as in Example 1. The enzyme was tested for thermal stability after incubation at 37°C for 72 hours and for crude enzyme activity at 50°C to exclude variants with decreased activity compared to wild-type LreuglsA. The total volume of the glutaminase activity assay system was 1 mL. 880 μL of a 200 mM glutamine solution was preheated in a 50°C water bath for 5 minutes. 20 μL of the crude enzyme solution was quickly added and mixed. For the control, 100 μL of a 15% trichloroacetic acid solution was quickly added and mixed. The reaction was allowed to react at 50°C for 5 minutes. 100 μL of a 15% trichloroacetic acid solution was quickly added and mixed. The reaction was terminated by the addition of 100 μL of the solution at 15% trichloroacetic acid.

[0055] The results are as follows Figure 2 、 Figure 3 As shown, the enzyme activities of the N2W and T21D mutants were 50% lower than those of the wild type. After incubation at 37°C for 72 hours, the relative enzyme activity of the H105K mutant was 247.36% of that of the wild type. Comparison of the RMSF values ​​of the H105K mutant and wild type during a 30 ns MD simulation at 37°C revealed that H105K has less structural flexibility than the wild type, resulting in its excellent thermal stability. Furthermore, an energy-based ddG of -1.93 kJ / mol confirms this possibility.

[0056] In a 1000 μL reaction system (calculated at final concentration), containing 200 g / L NaCl, 200 mM glutamine, and 50 mM Tris-HCl buffer, the enzyme solution was added and reacted at 50°C for 5 minutes. The enzyme activity was determined to verify the salt tolerance of the H105K mutant. The relative enzyme activity under 20% NaCl reached more than 85% of that in a NaCl-free environment, which is equivalent to the salt tolerance of the wild type, indicating that the mutation did not affect the salt tolerance of LreuglsA.

[0057] Table 1 Specific enzyme activity / enzyme activity stability of different mutants

[0058]

[0059]

[0060] Note: Stability after 72h incubation refers to the ratio of enzyme activity after 72h incubation to the enzyme activity after 72h incubation with wild type enzyme (WT).

[0061] Example 4: Application of mutant strains

[0062] The Bacillus subtilis expressing mutant H105K constructed in Example 1 or the enzyme solution obtained by disrupting the cells of the Bacillus subtilis was added to the soy sauce fermentation system. The results showed that the addition of the Bacillus subtilis containing the mutant or the enzyme solution obtained by disrupting the cells significantly increased the glutamate content in the fermented soy sauce.

[0063] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A glutaminase mutant, characterized in that Based on the glutaminase shown in SEQ ID NO.1, the histidine at position 105 is mutated to lysine.

2. A gene encoding the glutaminase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid is obtained by connecting the gene construction of claim 2 to the plasmid pMA5.

5. A recombinant microbial cell carrying the gene according to claim 2 or containing the recombinant plasmid according to claim 3.

6. A recombinant Bacillus subtilis, characterized in that Expressing the glutaminase mutant according to claim 1.

7. The recombinant Bacillus subtilis according to claim 6, characterized in that The Bacillus subtilis is Bacillus subtilis 168.

8. The method for preparing the glutaminase mutant according to claim 1, characterized in that: The recombinant Bacillus subtilis according to claim 6 or 7 is cultured in a culture medium for a period of time, and glutaminase in the cell culture fluid is collected.

9. Use of the recombinant Bacillus subtilis according to claim 6 or 7 as a cell catalyst, characterized in that: The application is to catalyze the hydrolysis of L-glutamine to L-glutamic acid.

10. Use of the glutaminase mutant according to claim 1 or the recombinant Bacillus subtilis according to any one of claims 6 to 7 in preparing food.

Images