Protein glutaminase with high activity on casein and soybean protein as well as production strain and application of protein glutaminase

Abstract

The invention discloses protein glutaminase as well as a production strain and application thereof. The preservation number of the Chryseobacterium lactis FET3 is CGMCC (China General Microbiological Culture Collection Center) NO: 33780, and the amino acid sequence of the produced protein glutaminase is as shown in SEQ ID NO. 4. The activity of the PG-producing enzyme of the Chryseobacterium lactis FET3 reaches 0.319 U / mL, the PG-producing enzyme shows unique catalytic advantages on casein and soybean protein substrates, and the solubility, foamability and emulsibility of protein can be remarkably improved; meanwhile, a double-peptone composite fermentation culture medium is innovatively developed, so that the enzyme production capacity of the FET3 strain is improved to 1.52 U / mL; in addition, the PG is purified by combining gradient low-temperature ethanol precipitation with an anion exchange chromatography technology, and finally the high-purity CL-PG enzyme is obtained. The invention provides an innovative technical scheme for efficient PG enzyme production and protein function improvement, and has important industrial application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a protein glutaminase with high activity against casein and soy protein, its producing strain, and its applications. Background Technology

[0002] Glutamine and asparagine residues in natural proteins cross-link with other amino acids through hydrogen bonds, leading to reduced protein solubility and consequently affecting their processing properties. Therefore, deamidation is crucial for improving protein functional properties. Currently, protein deamidation methods mainly include acid methods and enzymatic methods. While acid methods can significantly improve protein solubility, they easily lead to peptide chain hydrolysis and degradation of other amino acid residues, affecting protein flavor. In contrast, enzymatic deamidation has advantages such as mild reaction conditions and high specificity, and is gradually becoming the main method for protein modification. Protein-glutaminase (PG) is a deamidase with broad application prospects. It can specifically hydrolyze glutamine residues in proteins to generate L-glutamate and ammonia without destroying protein cross-links or hydrolysis, thus showing broad application prospects in the food industry. Casein, as the main protein in mammalian milk, is a typical representative of animal proteins and is widely used in dairy products, nutritional supplements, and food processing. Soy protein isolate (SPI) is a high-quality plant protein extracted from soybeans. Due to its high nutritional value and functionality, it occupies an important position in vegetarian food, artificial meat, beverages, and health foods.

[0003] However, currently, there are few strains capable of naturally producing PG. The strains reported to produce PG mainly include *Chryseobacterium proteolyticum*, *Chryseobacterium gleum*, and *Chryseobacterium indologenes*, which generally suffer from low enzyme activity and significant differences in efficacy against different protein substrates, lacking substrate universality. Existing protein glutaminases exhibit low catalytic deamidation efficiency on casein or soy protein isolate. *Chryseobacterium proteolyticum* is currently the only strain approved for industrial production, but its enzyme production capacity is low, with an enzyme activity of approximately 0.21 U / mL. Therefore, developing a novel protein glutaminase that exhibits significant and balanced high efficiency against both representative animal proteins (casein) and plant proteins (soy protein isolate) is of significant industrial value for broadening its application in diverse protein matrices, improving product quality, and developing novel functional foods. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a strain of *Lactobacillus lactis* that produces protein glutaminase and its applications.

[0005] Another objective of this invention is to provide a novel protein glutaminase, its encoding gene, and its applications.

[0006] Another object of the present invention is to provide a method for preparing the protein glutaminase.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A *Chryseobacterium lactis* FET3-CL, which produces protein glutaminase, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 33780 and deposit date of March 10, 2025.

[0009] The application of *Lactobacillus FET3-CL* described in this invention in the preparation of protein glutaminase.

[0010] A method for preparing protein glutaminase includes fermenting *Lactobacillus lactis* FET3-CL, collecting the supernatant to obtain a culture supernatant containing protein glutaminase. The fermentation medium is formulated as follows: 8-11 g tryptone, 3-5 g soybean peptone, 1.5-2.0 g fructose, 3-4 g KH₂PO₄, 3-5 g anhydrous MgSO₄, 0.8-1.5 g Leu, diluted to 1000 mL of water, and sterilized at 115°C for 30 min. A preferred fermentation medium formulation is: 11 g tryptone, 5 g soybean peptone, 1.5 g corn dextrin, 3.2 g KH₂PO₄, 3.9 g anhydrous MgSO₄, 1.1 g Leu, diluted to 1000 mL of water, and sterilized at 115°C for 30 min.

[0011] As a preferred embodiment of the present invention, the method further includes a step of extracting and purifying the protein glutaminase in the culture supernatant.

[0012] As a further preferred embodiment of the present invention, the extraction and purification includes the following steps:

[0013] Step 1: Collect the bacterial culture of Lactobacillus FET3-CL fermentation medium. Centrifuge the culture at 5000×g-8000×g for 10-30 min, discard the precipitate and collect the supernatant. Centrifuge the supernatant again at 8000×g-10000×g for 10-20 min to remove residual bacterial cells. Store at 4℃ for later use and record as L.

[0014] Step 2: The supernatant undergoes gradient low-temperature ethanol precipitation. The specific steps are as follows: First, add pre-cooled anhydrous ethanol solution to L to bring the total ethanol volume of the mixture to 50-60%. Precipitate slowly in an ice-water bath for 1-3 hours, then centrifuge at 8000×g-10000×g for 20 minutes to collect the supernatant L1 and remove the precipitate. This step removes a large amount of impurities. Next, add anhydrous ethanol solution to L1 again to bring the total ethanol volume of the mixture to 65%-75%. Precipitate slowly in an ice-water bath for 1-3 hours, then centrifuge at 8000×g-10000×g for 20 minutes. Collect the supernatant L2 and the precipitate separately. After drying the precipitate in a 30-60℃ oven to remove residual ethanol, reconstitute it with ultrapure water. This step is for the initial collection of CL-PG1 protein. Finally, add anhydrous ethanol solution to L2 again to bring the total ethanol volume of the mixture to 80%-90%. Precipitate slowly in an ice-water bath for 0.5-1 hour, then centrifuge at 8000×g-10000×g for 20 minutes. The supernatant L3 was removed and the precipitate was collected. The precipitate was dried in a 30-60℃ oven to remove residual ethanol and then reconstituted with ultrapure water. This step was to collect the remaining CL-PG2 protein. The purity of CL-PG2 protein was significantly higher than that of CL-PG1 protein, and the total enzyme activity recovery rate reached 45.6%.

[0015] Step 3: Replace the buffer system in CL-PG2 with PBS through an ultrafiltration tube, and further purify CL-PG2 using a cation exchange column to finally obtain CL-PG with high purity.

[0016] The protein glutaminase derived from *Chryseobacterium lactis* FET3-CL has the amino acid sequence shown in SEQ ID NO.4.

[0017] The gene encoding the protein glutaminase has a preferred nucleotide sequence as shown in SEQ ID NO.5.

[0018] A recombinant expression vector containing the gene encoding the protein glutaminase described above.

[0019] A genetically engineered bacterium, obtained by transfecting the recombinant expression vector with Escherichia coli as the host.

[0020] The application of the encoding gene, the recombinant expression vector, and the genetically engineered bacteria in the preparation of recombinant protein glutaminase.

[0021] The application of the protein glutaminase in deamidated plant protein.

[0022] A method for modifying plant protein using the protein glutaminase described in claim 5 includes fully hydrating the plant protein, adding purified CL-PG enzyme to it, and deamidating the plant protein isolate at 30-60°C for 1-4 hours.

[0023] The beneficial effects of this invention are:

[0024] (1) The CL strain obtained through screening exhibited a unique catalytic advantage for casein substrates. Substrate specificity assays showed that when using the synthetic substrate CBZ-Gln-Gly as a reference, its crude enzyme activity was defined as 100%; while when using natural casein and soy protein isolate as substrates, the relative enzyme activities were 152% and 65%, respectively. This characteristic contrasts sharply with the existing strain *Chryseobacterium proteolyticum*, whose relative enzyme activities for casein and soy protein isolate under the same reaction conditions (pH 7.0, 37℃) were only 42% and 22%, respectively. This indicates that the enzyme has a significant preference for the deamidation of glutamine residues in casein and soy protein isolate. This characteristic makes the CL strain a breakthrough application in the deep processing of dairy and soy products.

[0025] (2) By combining optimized enrichment medium with gradient screening strategy, the screening efficiency of high PG enzyme-producing strains was significantly improved. The enzyme activity of the sediment-derived Chrysobacterium FET3-CL reached 0.319 U / mL, which is 51.9% higher than that of the traditional Chrysobacterium glutamicum. At the same time, a novel double peptone (tryptone + soybean peptone) compound fermentation medium was developed, which increased the enzyme production capacity of the FET3-CL strain to 1.52 U / mL, which is 376.5% higher than that of the initial medium, laying the foundation for industrial production.

[0026] (3) By relying on the combination of genomics and heterologous expression technology, a novel PG enzyme gene (with only 83.65% similarity to known enzymes) was successfully identified and named CL-PG, which expanded the technical path for enzyme resource development.

[0027] (4) A highly efficient purification system was established by combining three-stage ethanol gradient precipitation (50-90%) with cation exchange chromatography, and the purity of the final product was improved to the level of a single band on electrophoresis. Figure 4 ).

[0028] (5) High-purity PG enzymes can be directly used for plant protein modification. Their specific catalytic ability can significantly improve the functional properties of plant proteins, providing a novel enzyme preparation solution for the food industry. The established strain screening-gene identification-heterologous expression technology system has universality and provides a standardized research paradigm for the development of other industrial enzymes. Attached Figure Description

[0029] Figure 1 A colony diagram of *Aureobacterium* after passage and stabilization;

[0030] Figure 2 This is a scanning electron microscope image of Chlorella vulgaris.

[0031] Figure 3 A diagram showing the protein differences between the novel CL-PG and deoxygenated PG;

[0032] Figure 4 The electrophoretic bands after CL-PG purification;

[0033] Figure 5 The specificity of CL-PG for different substrates;

[0034] Figure 6 To understand the specificity of PG enzyme (CP-PG) produced by Chlorella prionis for different substrates.

[0035] Information on the preservation of biological materials

[0036] FET3-CL, classified as Chryseobacterium lactis, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 33780, deposited on March 10, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto:

[0038] Example 1

[0039] A method for screening protein glutaminase strains, comprising the following steps:

[0040] Step 1: Collect orchard soil from different regions, and take 1.0 g of soil into a specially prepared 100 mL seed culture medium for rapid enrichment culture.

[0041] The seed culture medium consisted of: 10 g tryptone, 2 g yeast extract, 0.8 g anhydrous MgSO4, 1000 mL, sterilized at 115℃ for 30 min, pH 7.0; the culture conditions were: 30℃, 200 rpm, 4 h.

[0042] Step 2: Serially dilute the enriched bacterial culture to 10-1 2CFU / mL was spread onto LB medium containing 2.0% agarose and incubated at 30℃ for 3 days, with regular observation of colony status. Golden yellow, yellow, and pale yellow colony areas were selected using an inoculation loop and enriched in 25 mL of seed medium under the following conditions: 30℃, 200 rpm, 10 h. Golden yellow, yellow, and pale yellow colony areas were selected again for enrichment.

[0043] Step 3: Select the golden-yellow, yellow, and pale yellow single colonies that have been enriched multiple times in Step 2, and enrich and culture them again. Then, spread the amplified bacterial solution onto phenol red medium and incubate it at 30℃ for 2 days. The red single colonies are the suspected strains. The composition of phenol red medium is: 1.2% phenol red, 12 g tryptone, 1 g corn dextrin, 3 g KH2PO4, 4 g anhydrous MgSO4, 1.1 g Leu, 1.8% agarose, and make up to 1000 mL of water. Sterilize at 115℃ for 30 min.

[0044] Step 4: Select purple single colonies and place them in 25 mL of fermentation medium for culture. Continuously measure and record the PG enzyme activity in the medium and label the strains. The culture conditions are: 30℃, 200 rpm, 16 h. The fermentation medium formula is: 12 g tryptone, 1 g corn dextrin, 3 g KH2PO4, 4 g anhydrous MgSO4, 1.1 g Leu, diluted to 1000 mL of water, and sterilized at 115℃ for 30 min. The strain is continuously passaged, and after 5 generations, a stable CL-PG fermenting purified strain FET3-CL is obtained, with a CL-PG enzyme activity of 0.319 U / mL. Store at -80℃.

[0045] Under the same culture conditions, FET3-CL cultured in a new fermentation medium showed an increase in CL-PG enzyme activity to 1.52 U / mL, an increase of 376.5%. The fermentation medium was formulated as follows: 11 g tryptone, 5 g soybean peptone, 1.5 g corn dextrin, 3.2 g KH2PO4, 3.9 g anhydrous MgSO4, and 1.1 g Leu, diluted to 1000 mL of water and sterilized at 115°C for 30 min.

[0046] Morphological identification: The colony morphology of strain FET3-CL was observed by colony agar and scanning electron microscopy, as shown below. Figure 1 and Figure 2 As shown in the figure, the FET3-CL colonies are round, plump, golden yellow, with smooth edges and a sticky texture. The microscopic image shows that the bacteria are obviously rod-shaped with blunt ends and no obvious sharp ends. The surface is smooth without significant wrinkles or depressions and has uniform nanoscale granular texture.

[0047] Primer pairs (SEQ ID No. 1, SEQ ID No. 2) were designed to amplify the 16S rDNA of a high-yielding protein glutaminase strain. The wild-type strain's 16S rDNA was amplified, and the amplified gene was sent to a company for sequencing, as shown in SEQ ID No. 3. A search of the NCBI nucleic acid database revealed that the full-length 16S rDNA sequence of the *Chryseobacterium lactis* strain showed 98.34% similarity to the 16S rDNA sequences of all *Chryseobacterium lactis* strains in the database. Therefore, based on 16S rDNA sequence analysis, the strain was preliminarily identified as *Chryseobacterium lactis*.

[0048] Example 2

[0049] A method for determining the enzyme activity of protein glutaminase, comprising the following steps:

[0050] Determination of the ammonia standard curve: Weigh 0.314 g of ammonium chloride, dissolve it in water, and bring the volume to 1000 mL. Then, add 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of the solution to test tubes, and add water to 10 mL, so that the final concentrations in each test tube are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μg / mL, respectively. Pipette 12 μL of the ammonium chloride solution into a 96-well plate, add 48 μL of water, mix well, then add 60 μL of colorimetric reagent A, mix well, add 30 μL of colorimetric reagent B, mix well, and then add 60 μL of colorimetric reagent C, mix well, incubate at 37℃ for 20 min, cool under running water, and measure the OD value at OD630 with the ammonia concentration of 0 as the reference. Plot the OD630-ammonia concentration curve to obtain the ammonia standard curve.

[0051] PG enzyme activity assay: Using Cbz-Gln-Gly, casein, or soy protein isolate as substrates, the supernatant was diluted 10-fold. 0.1 mL of the diluted supernatant was added to a test tube, followed by 1 mL of the same mass ratio of Cbz-Gln-Gly, casein, or soy protein isolate. The reaction was carried out in a 37°C water bath for 30 min. Then, 1 mL of trichloroacetic acid solution was added, mixed thoroughly, and the reaction was terminated. The ammonia content in the solution after the reaction was determined using the phenol method. The absorbance value (A1) was measured at OD630. The control group was prepared by adding trichloroacetic acid solution first, reacting for 30 min, and then adding the substrate solution. The remaining procedures were the same as the experimental group. The OD630 value was measured as A2. One enzyme activity unit (IU) is defined as the amount of enzyme required to produce 1 μmol of ammonia per minute. The formula for calculating enzyme activity in the fermentation broth is as follows:

[0052] Enzyme activity (U / mL) = (A1 - A2) × 21 × n / 30 / 17.03 / a

[0053] A1: Absorbance of the experimental group;

[0054] A2: Absorbance of the control group;

[0055] a: The slope of the standard curve for ammonia determination by the phenol method.

[0056] The specificity of CL-PG and PG enzyme produced by *Chlorella vulgaris* (CP-PG) for different substrates was investigated. Results are shown in [Figure number missing]. Figure 5 and Figure 6 As shown in the figure, for CL-PG, casein is the best substrate with a relative enzyme activity of 152%, followed by Cbz-Gln-Gly and soy protein isolate, with relative enzyme activities of 100% and 65%, respectively. For CP-PG, Cbz-Gln-Gly is the best substrate with a relative enzyme activity of 100%, followed by casein and wheat gliadin, with relative enzyme activities of 42% and 30%, respectively, while the relative enzyme activity of soy protein isolate is only 22%.

[0057] Example 3

[0058] Genome-wide genome sequencing and heterologous expression validation of the PG gene in *Cyclocarya paliurus* strains:

[0059] Extraction of bacterial genomic DNA: Collect 1.0 × 10⁹ bacterial cultures (OD600 of 1-1.5 per mL) in a 2 mL centrifuge tube, centrifuge at 12,000 × g for 30 s, and discard the supernatant. Resuspend the precipitate in 150 μL of Buffer S containing RNase A. Add 20 μL of lysozyme stock solution, mix well, and incubate at room temperature for 5 min. Add 30 μL of 0.25 M EDTA (pH 8.0), mix well, and incubate on ice for 5 min. Add 450 μL of Buffer GA, vortex for 15 s, and incubate at 65 °C for 10 min. Add 400 μL of Buffer GB and 1 mL of Buffer DV (pre-chilled at 4 °C), mix vigorously, and centrifuge at 12,000 × g for 2 min. Discard the upper phase as much as possible, retaining the interphase precipitate and the lower phase. Add 1 mL of Buffer DV (pre-chilled at 4 °C), mix vigorously, and centrifuge at 12,000 × g for 2 min. Discard the upper phase and transfer the lower phase to a filter (place the filter in a 2 mL centrifuge tube). Centrifuge at 12,000 × g for 1 min. Discard the filter and add 400 μL of Buffer BV to the filtrate, mixing thoroughly. Place the preparation tube in a 2 mL centrifuge tube, transfer the above mixture into the preparation tube, and centrifuge at 12,000 × g for 1 min. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 500 μL of Buffer W1, and centrifuge at 12,000 × g for 1 min. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, add 700 μL of Buffer W2, and centrifuge at 12,000 × g for 1 min. Wash once more with 700 μL of Buffer W2 using the same method. Discard the filtrate, return the preparation tube to the original 2 mL centrifuge tube, and centrifuge at 12,000 × g for 1 min. Place the preparation tube into another clean 1.5 mL centrifuge tube, add 100-200 μL of Eluent or deionized water to the center of the silica membrane, and let stand at room temperature for 1 min. Centrifuge at 12,000×g for 1 min to elute the DNA.

[0060] The obtained DNA was sent to a genetic engineering company for whole-genome analysis. The gene sequences of *Chlorella vulgaris* were compared to analyze potential PG genes. The obtained PG protein was compared with that of *Chlorella vulgaris* prion. Figure 3The similarity was 83.65%. Upstream and downstream primers (SEQ ID NO.6 and SEQ ID NO.7) were designed based on the PG gene. Homologous arms of the PET-28a vector were designed using Snapgene software for PCR amplification. The amplification program was: 98℃ for 5 min pre-denaturation, 98℃ for 30 s denaturation, 55℃ for 30 s annealing, 72℃ for 60 s extension, and 72℃ for 10 min final extension, for 30 cycles. The obtained PCR product was verified by nucleic acid electrophoresis and purified. Finally, an engineered *E. coli* strain was obtained using a one-step cloning kit. Heterologous expression of the CL-PG gene was induced by culturing at 37℃ and using 0.5 mM IPTG. CL-PG protein was collected by cell lysis and purified by nickel column chromatography. The enzyme activity of CL-PG after activation was 10.56 U / mg.

[0061] Example 4

[0062] A CL-PG gradient purification process includes the following steps: collecting bacterial culture from FET3-CL fermentation medium, centrifuging the bacterial culture at 8000×g for 20 min, discarding the precipitate and collecting the supernatant; centrifuging the supernatant again at 10000×g for 20 min to remove residual bacterial cells, storing it at 4℃ for later use, denoted as L;

[0063] The supernatant was subjected to gradient low-temperature ethanol precipitation. The specific steps were as follows: First, pre-cooled anhydrous ethanol solution was added to L to bring the total ethanol volume of the mixture to 50%. The mixture was then slowly precipitated in an ice-water bath for 1 h, centrifuged at 10000×g for 20 min, and the supernatant L1 was collected to remove the precipitate. This step removed a large amount of impurities. Next, anhydrous ethanol solution was added to L1 again to bring the total ethanol volume of the mixture to 70%. The mixture was then slowly precipitated in an ice-water bath for 1 h, centrifuged at 10000×g for 20 min, and the supernatant L2 and the precipitate were collected separately. The precipitate was dried in a 50℃ oven to remove residual ethanol and then reconstituted with ultrapure water. This step was for the initial collection of CL-PG1 protein. Finally, anhydrous ethanol solution was added to L2 again to bring the total ethanol volume of the mixture to 80%. The mixture was then slowly precipitated in an ice-water bath for 1 h. Centrifuge at 10000×g for 20 min to remove the supernatant (L3) and collect the precipitate. After removing residual ethanol by drying in a 50℃ oven, the precipitate is reconstituted with ultrapure water. This step is to collect the remaining CL-PG2 protein. The purity of CL-PG2 protein is significantly higher than that of CL-PG1 protein, and the total enzyme activity recovery rate reaches 45.6%.

[0064] Step 3: Replace the buffer system in CL-PG2 with PBS using an ultrafiltration tube, adjusting the pH to 7. Since CL-PG cannot be adsorbed by the anion exchange column at this pH and will be eluted, impurities will be adsorbed. Therefore, CL-PG2 is further purified using a cation exchange column, ultimately obtaining highly pure CL-PG, which appears as a single band, as shown below. Figure 4 As shown.

[0065] Example 5

[0066] A method for modifying plant protein with CL-PG: Soy protein isolate prepared in advance was placed in pure water at a concentration of 1%, magnetically stirred at room temperature for 2 hours, and then hydrated overnight in a refrigerator. Purified CL-PG enzyme was added at a dosage of 5 U / g, and the mixture was placed in a 50℃ water bath for deamidation modification of the soy protein isolate. Modification times were 1, 2, 3, and 4 hours. After modification, the mixture was placed in an 80℃ water bath for 10 min to inactivate the CL-PG enzyme, followed by rapid cooling in ice water. The solubility, foaming properties, and emulsification characteristics of the protein were then measured.

[0067] Properties of soy protein isolate after deamidation:

[0068] The solubility of soy protein isolate increased from 14% to a maximum of 31%; turbidity decreased from 9.5% to a maximum of 3.6%; emulsifying properties increased from 17.5% to a maximum of 32%; and foaming properties increased from 149% to a maximum of 249%. The degree of deamidation of the protein increased to 30%, but the degree of hydrolysis remained below 4.5%. These results indicate that CL-PG only affects the amide groups of the protein and can significantly improve the properties of soy protein isolate.(The methods for determining solubility, emulsifying properties, and degree of deamidation are referenced in Yong, YH; Yamaguchi, S.; Matsumura, Y. Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten. J. Agric. Food Chem. 2006, 54, 6034–6040. The method for determining turbidity is referenced in Hu, Y.; Sun-Waterhouse, D.; Liu, P.; Cui, C.; Wang, W. Modification of rice protein with glutaminase for improved structural and sensory properties. International Journal of Food Science & Technology. 2019, 54(7), 2458–2467. The method for determining foaming properties is referenced in Kanu, PJ; Kanu, JB; Sandy, EH; Kandeh, JBA; Mornya, PMP; Huiming, Z. Optimization of enzymatic hydrolysis of defatted sesame flour.) bydeifferent proteases and their effect on the functional properties of theresulting protein hydrolysate. Am. J. Food Technol. 2009, 4 (6), 226–240. For the method of determining the degree of hydrolysis, please refer to PM Nielsen, D. Petersen, C. Dambmann. Improved Method forDeterminingFood Protein Degree of Hydrolysis. Journal of Food Science. 2001,66(5), 642-646.).

Claims

1. A type of *Lactobacillus* that produces protein glutaminase (… Chryseobacterium lactis FET3-CL, characterized in that, It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 33780 and deposit date of March 10, 2025.

2. The application of the *Lactobacillus FET3-CL* as described in claim 1 in the preparation of protein glutaminase.

3. A method for preparing protein glutaminase, characterized in that, The method includes fermenting and culturing *Lactobacillus lactis* FET3-CL as described in claim 1, collecting the supernatant to obtain a culture supernatant containing protein glutaminase. The fermentation medium is formulated as follows: 8-11 g tryptone, 3-5 g soybean peptone, 1.5-2.0 g fructose, 3-4 g KH₂PO₄, 3-5 g anhydrous MgSO₄, 0.8-1.5 g Leu, diluted to 1000 mL of water, and sterilized at 115°C for 30 min. A preferred fermentation medium formulation is: 11 g tryptone, 5 g soybean peptone, 1.5 g corn dextrin, 3.2 g KH₂PO₄, 3.9 g anhydrous MgSO₄, 1.1 g Leu, diluted to 1000 mL of water, and sterilized at 115°C for 30 min.

4. The method according to claim 3, characterized in that, It also includes a step of extracting and purifying the protein glutaminase in the culture supernatant.

5. Derived from the *Lactobacillus* of claim 1 ( Chryseobacterium lactis FET3-CL protein glutaminase, characterized in that, The amino acid sequence is shown in SEQ ID NO.

4.

6. The gene encoding the protein glutaminase of claim 5, preferably with the nucleotide sequence shown in SEQ ID NO.

5.

7. A recombinant expression vector containing the gene encoding the protein glutaminase described in claim 5.

8. A genetically engineered bacterium, characterized in that, The recombinant expression vector described in claim 7 was obtained by transfecting Escherichia coli with the host.

9. The use of the encoding gene of claim 6, the recombinant expression vector of claim 7, and the genetically engineered bacteria of claim 8 in the preparation of recombinant protein glutaminase.

10. The application of the protein glutaminase according to claim 5 in deamidated plant protein.

11. A method for modifying plant protein using the protein glutaminase described in claim 5, characterized in that... This involves fully hydrating the plant protein, adding purified protein glutaminase, and deamidating the plant protein isolate at 30-60℃ for 1-4 hours.

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