L-glutaminase with high catalytic activity

Abstract

The invention discloses L-glutaminase with high catalytic activity, which can effectively increase the content of L-glutamic acid in a soy sauce fermentation simulation system, and belongs to the technical field of biology. The invention discloses an ancestor L-glutaminase sequence capable of increasing the content of L-glutamic acid in a soy sauce fermentation simulation system, and belongs to the technical field of biology. The ancestor L-glutaminase disclosed by the invention can be used for effectively increasing the content of L-glutamic acid in a high-temperature, low-pH and high-salt environment. The L-glutaminase disclosed by the invention has a relatively good application prospect in soy sauce fermentation.

Description

Technical Field

[0001] The invention relates to L-glutaminase with high catalytic activity, belonging to the field of biotechnology. Background Art

[0002] L-glutaminase (EC 3.5.1.2) is an amidohydrolase that catalyzes the hydrolysis of the γ-amide bond of L-glutamine into L-glutamic acid and free ammonia, and is widely distributed in prokaryotes and eukaryotes. In addition, some L-glutaminases also have the ability to transfer γ-glutamyl groups, which can catalyze the transfer reaction of hydroxylamine, methylamine and ethylamine as acceptor molecules. L-glutaminases have great application potential in the fields of food and medicine. In the food industry, L-glutaminases can be used to enhance the flavor of soy sauce, synthesize theanine, improve the flavor of fermented foods and protein hydrolysates, etc. In the field of medicine, L-glutaminases can be used for enzyme therapy of cancer, and also have antiviral, antibacterial and antioxidant activities. In addition, a biosensor for detecting L-glutamine levels has been developed based on L-glutaminases, which has good prospects for clinical analysis applications. Therefore, it is very necessary to explore highly active L-glutaminases, which has become a current research hotspot.

[0003] At present, researchers have fully studied L-glutaminases from Aspergillus oryzae, Bacillus, Pseudomonas, Micrococcus luteus, Zygosaccharomyces rouxii, Cryptococcus, etc. According to current research, the highest specific activity of actinomycete L-glutaminases recorded is 132.2U / mg from Streptomyces canarius, the highest specific activity of other bacterial L-glutaminases recorded is 1230U / mg from Micrococcus luteus K-3, the highest specific activity of yeast L-glutaminases recorded is 1065U / mg from Debaryomyces spp., and the highest specific activity of other fungal L-glutaminases recorded is 869U / mg from Penicillium brevicompactum NRC 829. The fermentation of soy sauce is often carried out in an environment of high temperature (40-55°C), acidic (pH 5.0-7.0) and high salt concentration (13-20%). The existing L-glutaminase generally has low enzyme activity, so more methods are needed to explore and discover more new enzymes with excellent enzymatic properties as candidate enzymes for soy sauce fermentation. Summary of the invention

[0004] In order to address the deficiencies in the prior art, the present invention obtains L-glutaminase protein with high specific enzyme activity through ancestral sequence reconstruction. The protein obtained by the present invention does not come from proteins that already exist in nature, but is obtained by the ancestral sequence reconstruction method. The ancestral enzyme protein obtained by the present invention has good application prospects in soy sauce fermentation.

[0005] The present invention provides an L-glutaminase protein, wherein the L-glutaminase protein is as shown in (a) or (b):

[0006] (a) L-glutaminase protein Anc165 having an amino acid sequence as shown in SEQ ID NO.1;

[0007] (b) L-glutaminase protein Anc194 having an amino acid sequence as shown in SEQ ID NO.2.

[0008] The present invention also provides a gene encoding the L-glutaminase protein.

[0009] In one embodiment, the gene encoding the L-glutaminase protein Anc165 has a nucleotide sequence shown in SEQ ID NO.3.

[0010] In one embodiment, the gene encoding the L-glutaminase protein Anc194 has a nucleotide sequence shown in SEQ ID NO.4.

[0011] The invention also provides a recombinant plasmid carrying the gene.

[0012] The present invention also provides a recombinant cell expressing the L-glutaminase protein.

[0013] In one embodiment, the recombinant cell is hosted by Escherichia coli.

[0014] In one embodiment, the recombinant cell uses Escherichia coli BL21 (DE3) as a host and a pET series plasmid as an expression vector.

[0015] The present invention also provides a method for producing L-glutaminase protein, the method comprising:

[0016] (1) inoculating the recombinant Escherichia coli into a culture medium and adding IPTG for induction culture;

[0017] (2) Collecting the fermentation liquid obtained in step (1), separating and obtaining cells, and disrupting the cells to obtain L-glutaminase protein.

[0018] In one embodiment, the culture temperature of step (1) is between 30 and 37°C.

[0019] In one embodiment, step (1) is to culture the recombinant Escherichia coli to an OD of 0.6-1.0.

[0020] In one embodiment, the induction temperature of step (1) is 15-30°C, preferably 20°C.

[0021] In one embodiment, the induction time in step (1) is 10-36 hours, preferably 32 hours.

[0022] In one embodiment, the culture medium in step (1) includes but is not limited to LB medium.

[0023] In one embodiment, the final concentration of IPTG in step (1) is 0.01-0.5 mmol / L; preferably, the IPTG concentration is 0.1 mmol / L.

[0024] In one embodiment, the induction condition of step (1) is preferably induced at 20°C for 32 hours.

[0025] In one embodiment, the crushing in step (2) is preferably high-pressure homogeneous crushing.

[0026] In one embodiment, the L-glutaminase protein obtained by the above method is purified, and the purification is Ni 2+ -Sepharose affinity column purification.

[0027] The present invention also provides a method for increasing the content of L-glutamic acid in soy sauce, which comprises adding the L-glutaminase protein into a soy sauce fermentation system to hydrolyze L-glutamine to generate L-glutamic acid.

[0028] In one embodiment, the soy sauce fermentation system uses isolated soy protein as raw material.

[0029] In one embodiment, the soy sauce fermentation system uses soy protein isolate as raw material, and pre-treats the soy protein isolate, wherein the pre-treatment includes enzymolysis of the soy protein isolate using alkaline protease and compound flavor enzyme.

[0030] In one embodiment, the hydrolysis is carried out at a temperature of 50-65° C. and a pH of 7.0-9.0 for at least 30 min.

[0031] In one embodiment, the reaction time is 10 min to 2 h.

[0032] In one embodiment, the hydrolysis is carried out at 55° C., pH 6.0, and a final NaCl concentration of 15%.

[0033] The present invention also provides application of the L-glutaminase protein, the recombinant cell, or the method in soy sauce fermentation.

[0034] Beneficial effects:

[0035] (1) The present invention reconstructs the ancestral sequence to obtain new L-glutaminase proteins Anc165 and Anc194 with amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0036] (2) The L-glutaminase protein constructed by the present invention can effectively increase the content of L-glutamic acid in the reaction system. After adding the L-glutaminase protein of the present invention, the content of L-glutamic acid can be increased by 12.5% ​​and 5% respectively compared with the reaction system without adding the enzyme protein.

[0037] (3) The L-glutaminase protein obtained by the present invention shows good specific enzyme activity, can adapt to the soy sauce fermentation environment, can effectively hydrolyze L-glutamine into L-glutamic acid, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 SDS protein electrophoresis of ancestral L-glutaminase Anc165 and Anc194.

[0039] Figure 2 The specific enzyme activities of Anc165 and Anc194 at 55°C and pH 6.0.

[0040] Figure 3 Optimal temperature, pH value, thermal stability and salt tolerance of the ancestral L-glutaminase; (a) Optimal reaction temperature of Anc165 and Anc194; (b) Optimal reaction pH value of Anc165 and Anc194; (c) Thermal stability of Anc165 and Anc194; (d) Salt tolerance of Anc165 and Anc194.

[0041] Figure 4 The curve of L-glutamic acid content in the simulated fermentation system of soy sauce at 55°C, pH 6.0, and final concentration of 15% NaCl. The addition amount of each ancestral L-glutaminase is 0.05 mg. Control is the reaction system without adding ancestral L-glutaminase, and the others correspond to ancestral L-glutaminase Anc165 and Anc194 respectively. DETAILED DESCRIPTION

[0042] Construction of soy sauce fermentation simulation system:

[0043] 0.5g soy protein isolate was dissolved in 50mM Tris-HCl buffer (pH 8.0), fixed to 5mL, and then 500μL alkaline protease was added. After 2 hours in a 60℃ water bath, 500μL compound flavor enzyme was added, and the water bath was continued at 50℃ for 4 hours. After the end, the protease activity was terminated by 90℃ for 10 minutes in a water bath. The simulation system was centrifuged at 4℃ and 10000rpm for 20 minutes, and the upper layer sample was collected and fixed to 5mL for use as a substrate for the bioconversion reaction.

[0044] The α-amino acid content was determined using a free α-amino acid (FAAA) enzyme-linked immunosorbent assay (ELISA) kit to detect the degree of enzymatic hydrolysis of soy protein isolate. NaCl was added to the upper sample to a final concentration of 15% and the pH was adjusted to 6.0. The prepared pure enzyme solution (0.05 mg) was added to the simulation system, kept warm in a 55°C water bath, 200 μL of the reaction solution was taken out at different time points, and the enzyme reaction was terminated at 90°C for 10 minutes. The content of L-glutamic acid was detected using an L-glutamic acid (L-GLU) ELISA detection kit.

[0045] Enzyme activity determination method (with L-glutamine as substrate):

[0046] The activity of the ancestral L-glutaminase was determined by the Nessler reagent method. The substrate (74mM L-glutamine) was prepared with Na2HPO4-C6H8O7 buffer (100mM, pH6.0), 1100μL of the substrate was incubated at 55℃ for 10 minutes, 100μL of the enzyme solution diluted to an appropriate multiple was added, and 100μL of the terminator (1.5M trichloroacetic acid) was added to the 1100μL substrate incubated at 55℃ for 10 minutes to terminate the reaction. The mixture of the above 100μL enzyme solution and 100μL terminator was added to the 1100μL substrate incubated at 55℃ for 10 minutes, and the mixture was incubated at 55℃ for 10 minutes. The resulting solution was used as the control for absorbance detection. After the reaction, centrifuge at 12000 rpm for 2 minutes, take 100 μL of supernatant, add 3400 μL of deionized water and 500 μL of Nessler reagent (a mixed solution of 0.09 mol / L K2HgI4 and 2.5 mol / L KOH), mix well and measure the absorbance at a wavelength of 436 nm.

[0047] Enzyme activity is defined as the amount of enzyme required to catalyze L-glutamine to produce 1 μmol NH3 per minute, which is defined as one unit of enzyme activity.

[0048] Relative enzyme activity: The relative enzyme activity at the end of the reaction under the optimal reaction conditions is A0, and the relative enzyme activity measured at the end of the reaction under other reaction conditions is A1. A1 / A0 is the relative enzyme activity.

[0049] Residual enzyme activity (%): The specific enzyme activity of the unincubated enzyme measured at 55°C and pH 6.0 is A0, and the specific enzyme activity measured at the end of the reaction after incubation is A1. A1 / A0 is the residual enzyme activity.

[0050] Example 1: Construction of ancestral L-glutaminase expression strain

[0051] Based on the codon preference of E. coli, the ancestral L-glutaminase gene sequence (the nucleotide sequences of the ancestral L-glutaminase Anc165 and Anc194 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively) was synthesized and cloned into the NcoⅠ-XhoⅠ restriction site of the pET-28(a) vector to obtain the corresponding expression plasmid. The obtained expression plasmids were transformed into E. coli BL21(DE3) respectively, and the correct ones were verified to be recombinant E. coli expressing the ancestral L-glutaminase.

[0052] Example 2: Expression and purification of ancestral L-glutaminase protein

[0053] The recombinant E. coli constructed in Example 1 was inoculated into LB medium containing 100 μg / mL kanamycin sulfate and cultured at 37°C and 220 rpm for 10 h. The resulting culture solution was transferred to TB medium (100 μg / mL kanamycin sulfate) at a 2% inoculum (v / v) and cultured at 37°C and 220 rpm until OD 600 About 0.8; add LIPTG with a final concentration of 0.1mmol / LIPTG, and continue fermentation at 20℃ and 220rpm for 32h.

[0054] The cells obtained by the above method were collected by centrifugation, resuspended in Tris-HCl buffer (50 mM, pH 8.0) at 4°C and disrupted by high pressure homogenization. After the cells were disrupted, the supernatant was collected by centrifugation at 10,000 rpm at 4°C for 20 min and then purified by Ni 2+ -Sepharose affinity column (cytiva) to purify the protein. The purity of the purified protein was analyzed by SDS-PAGE. The results are shown in Figure 1 As shown, Anc165 and Anc194 were both expressed in a soluble form.

[0055] The following reaction system was prepared: a 74 mM L-glutamine solution was prepared with Na2HPO4-C6H8O7 buffer (100 mM, pH 6.0), and purified Anc165 protein and Anc194 were added respectively. The reaction was carried out at 55°C for ten minutes, and the specific enzyme activities of Anc165 and Anc194 were detected. Figure 2 As shown, the specific enzyme activities of L-glutaminase Anc165 and Anc194 were 4384 U / mg and 1221 U / mg, respectively.

[0056] Example 3: Characterization of the enzymatic properties of ancestral L-glutaminase

[0057] According to the enzyme activity reaction system of Example 2, the relative enzyme activities of Anc165 and Anc194 were detected under different temperature and pH conditions to determine the optimal reaction temperature and the optimal reaction pH. Figure 3 As shown, the relative enzyme activity of L-glutaminase Anc165 can reach more than 76% at 50-60°C, and the relative enzyme activity can reach more than 81% in an environment of pH 6-8; the relative enzyme activity of L-glutaminase Anc194 can reach more than 62% at a temperature of 50-70°C, and the relative enzyme activity can reach more than 73% in an environment of pH 6-8.

[0058] The thermal stability was determined by incubating the enzyme protein prepared in Example 2 at 55, 60, 65, and 70° C. for 30 min, and then measuring the specific enzyme activity at 55° C. and pH 6.0 to calculate the residual enzyme activity.

[0059] The salt tolerance test was performed by incubating the enzyme protein prepared in Example 2 in a 15% NaCl solution at 55° C. for 5 min. The specific enzyme activity was then measured at 55° C. and pH 6.0, and the residual enzyme activity was calculated.

[0060] The results are as follows Figure 3 As shown, the residual enzyme activity of L-glutaminase Anc165 after incubation at 55-65°C for 30 minutes remained above 96%; the residual enzyme activity of L-glutaminase Anc194 after incubation at 55-60°C for 30 minutes remained above 70%. The residual enzyme activity of L-glutaminase Anc165 remained at 100% in the environment of 15% NaCl.

[0061] Example 4: Application of ancestral L-glutaminase in soy sauce fermentation simulation system

[0062] 0.5g of soy protein isolate (purchased from Shanghai Yuanye, China) was dissolved in 50mM Tris-HCl buffer (pH 8.0), and the volume was adjusted to 5mL. Then 500μL of alkaline protease (enzyme activity of 400U / mL) was added. After 2 hours of water bath at 60℃, 500μL of compound flavor enzyme (enzyme activity of 40U / mL) was added, and the water bath was continued at 50℃ for 4 hours. After the end, the protease activity was terminated by water bath at 90℃ for 10 minutes. The simulation system was centrifuged at 4℃ and 10000rpm for 20 minutes, and the upper layer sample was collected and adjusted to 5mL for use as a substrate for the biotransformation reaction.

[0063] The α-amino acid content was determined using a free α-amino acid (FAAA) enzyme-linked immunosorbent assay (ELISA) kit to detect the degree of enzymatic hydrolysis of soy protein isolate. NaCl was added to the upper sample to a final concentration of 15%, and the pH was adjusted to 6.0. The pure enzyme solutions (0.05 mg) of Anc165 and Anc194 prepared in Example 1 were added to the simulation system, respectively, and kept warm in a 55°C water bath. 200 μL of the reaction solution was taken out at different time points, and the enzyme reaction was terminated at 90°C for 10 minutes. The content of L-glutamic acid was detected using an L-glutamic acid (L-GLU) ELISA detection kit.

[0064] After the soy protein isolate was enzymatically hydrolyzed by alkaline protease and compound flavor enzymes (6h in total), the test results showed that the α-amino acid content in the reaction supernatant reached 1146ng / mL, indicating that the soy protein isolate was successfully enzymatically hydrolyzed. Afterwards, NaCl was added to 5mL of the supernatant after the reaction to make the final concentration of NaCl 15%, and the pH was adjusted to 6.0. Then, 500μL of ancestral L-glutaminase Anc165 or Anc194 with a concentration of 0.1mg / mL was added and reacted at 55℃ to determine the content of L-glutamate. The supernatant without L-glutaminase was used as a control and treated at the same temperature for the same time. During the initial 1h reaction, the L-glutamate content in the three cases increased rapidly over time, and Anc165 showed a higher L-glutamate accumulation rate than the other two cases. Thereafter, the accumulation rate of L-glutamate slowed down until the end of the reaction, because high temperature, low pH and high salt environment affect the activity of L-glutaminase. As the reaction time increased, L-glutaminase gradually lost its activity. Finally, the L-glutamate content produced by the system with Anc165 added at 2 hours increased by 12.5% ​​compared with the system without Anc165, and the L-glutamate content produced by the system with Anc194 added at 2 hours increased by 5% compared with the system without Anc194.

[0065] The above results indicate that the L-glutaminases Anc165 and Anc194 obtained by ancestral sequence reconstruction in the present invention can effectively increase the content of L-glutamate in the soy sauce fermentation simulation system under high temperature, low pH and high salt environment.

[0066] Comparative Example:

[0067] The specific implementation method is the same as Example 4, except that L-glutaminase YbgJ (Gene ID: 938416) from Bacillus subtilis 168 in the prior art is used in the soy sauce fermentation simulation system, and the L-glutamate content is detected under the same conditions. The results show that the L-glutaminase content produced by the system with the addition of L-glutaminase in the prior art at 2 hours is 2.6% higher than that of the system without the addition.

[0068] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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. L-glutaminase, characterized in that As shown in (a) or (b): (a) L-glutaminase protein Anc165 having an amino acid sequence as shown in SEQ ID NO.1; (b) L-glutaminase protein Anc194 having an amino acid sequence as shown in SEQ ID NO.

2.

2. A gene encoding the L-glutaminase according to claim 1.

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

4. A recombinant cell expressing the L-glutaminase protein according to claim 1.

5. A recombinant Escherichia coli, characterized in that The L-glutaminase according to claim 1 is expressed by using Escherichia coli BL21 (DE3) as a host and pET series plasmids as expression vectors.

6. A method for producing L-glutaminase, characterized in that: The method comprises: (1) culturing the recombinant Escherichia coli according to claim 5 in a culture medium, and adding IPTG to induce the expression of the L-glutaminase; (2) Collecting L-glutaminase protein in the fermentation broth obtained in step (1).

7. A method for increasing the content of L-glutamic acid in soy sauce, characterized in that: The L-glutaminase according to claim 1 is added into the soy sauce fermentation system to hydrolyze L-glutamine to generate L-glutamic acid.

8. The method according to claim 7, characterized in that The hydrolysis is carried out at a temperature of 50-65° C. and a pH of 7.0-9.0 for at least 30 minutes.

9. A method for hydrolyzing L-glutamine in soy sauce fermentation raw materials, characterized in that: The L-glutaminase according to claim 1 is contacted with a soy sauce fermentation raw material.

10. Use of the L-glutaminase according to claim 1, or the recombinant cell according to claim 4, or the recombinant Escherichia coli according to claim 5, or the method according to any one of claims 6 to 9 in the preparation of soy sauce.

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