Low-temperature neutral protease, method of producing same and use thereof

Abstract

The present application belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a low-temperature neutral protease from marine microorganism colwellia psychrerythraea, a preparation method thereof and application of the low-temperature neutral protease in hydrolysis of proteins. In the present application, a protease gene from colwellia psychrerythraea is connected with an expression vector pPIC9K to obtain a recombinant plasmid, then the recombinant plasmid is electrotransformed into Pichia pastoris cells, and a target gene is expressed by using methanol induction, and a centrifugal product is obtained after removing supernatant, thereby obtaining a recombinant low-temperature neutral protease. The recombinant low-temperature neutral protease can be used for hydrolysis of fish meal, and the recombinant low-temperature neutral protease has higher enzymatic activity in the range of 15 DEG C to 35 DEG C, and still maintains more than 60% at 15 DEG C, so that the recombinant low-temperature neutral protease is a neutral low-temperature metal protease which can maintain higher activity at low temperature or normal temperature.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a low-temperature neutral protease derived from the marine microorganism Colwellella horneri, its preparation method, and its application in protein hydrolysis. Background Technology

[0002] Protease (EC 3.4.21.14) is a general term for a class of enzymes that hydrolyze peptide bonds in proteins or peptides. Proteases are widely distributed in nature, primarily originating from molds, bacteria, or other microorganisms. Proteases have very important applications in food, medicine, textiles, leather tanning, detergents, cosmetics, animal and plant proteins, and waste treatment, including:

[0003] (1) Preparation of polypeptides: Proteases can act on protein macromolecules and hydrolyze them into small peptides or polypeptides of 2 to 20 amino acids. These oligopeptides produced by protease hydrolysis have different functions, but all have certain nutritional value and health benefits, and are widely used in food, medicine, feed and agriculture.

[0004] (2) Hydrolysis of anti-nutritional factors: The main protein-based anti-nutritional factors in food include trypsin inhibitors and antigenic protein lectins. Proteases can hydrolyze these proteins, reducing the amount of anti-nutritional factors in food.

[0005] (3) Meat tenderization: Proteases can also be used to tenderize meat. Neutral proteases can dissolve myofibrils and elastin, turning them into irregular structures, thus tenderizing the meat.

[0006] Currently, industrial proteases are mainly extracted from mesophilic microorganisms such as Aspergillus niger using solvent extraction. These enzymes generally exhibit high activity at 37–50°C, but their activity is extremely low below room temperature (25°C). Therefore, the search for low-temperature proteases is of great significance for their subsequent industrial applications.

[0007] Patent document CN104561238A discloses a method for screening strains producing low-temperature neutral protease. This method, through methods such as modifying the culture medium by adding marine natural substances, concentrating the sample to increase the microbial content per unit volume, and controlling screening conditions, screened strains producing low-temperature neutral protease from marine silt obtained at a water depth of approximately 600m, achieving a maximum crude enzyme activity of 35.5 U / ml. However, this screening method has low reproducibility and cannot be industrialized.

[0008] Patent document CN104004737A discloses a low-temperature protease from *Collimonas pratensis* JZB120004, along with related biomaterials and applications. This *Collimonas pratensis* JZB120004 produces proteases, chitinases, phosphatases, and heptaphiles, but no cellulase was detected. The extracellular protease from *Collimonas pratensis* JZB120004 has an optimal temperature of 30℃, exhibiting high activity within the 30℃–40℃ range. Above 40℃, its activity decreases sharply, but it remains above 70% at 10℃. The optimal pH is 7, with high activity within the pH range of 6.5–7.5, classifying it as a neutral, low-temperature metalloproteinase. Furthermore, the active substances from this *Collimonas pratensis* JZB120004 significantly inhibit the spread of peach brown rot lesions, making it suitable for the biological control of peach brown rot. Summary of the Invention

[0009] To address current industry needs, the purpose of this invention is to discover and develop proteases with high catalytic activity at low or room temperatures, suitable for preparing peptides and hydrolyzing large protein molecules. This invention provides an application of a neutral protease derived from marine microorganisms in the efficient hydrolysis of proteins, specifically an application of a low-temperature neutral protease derived from *Colwellia hornerae* in protein hydrolysis.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] This invention provides a low-temperature neutral protease (Chprotease), the amino acid sequence of which is shown in SEQ ID NO.2.

[0012] Furthermore, the nucleotide sequence of the low-temperature neutral protease (Chprotease) is shown in SEQ ID NO.1.

[0013] Furthermore, the present invention provides the low-temperature neutral protease (Chprotease) expression vector, wherein the nucleotide fragment encoding the low-temperature neutral protease described in SEQ ID NO.1 is inserted into the expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-Chprotease.

[0014] Furthermore, the present invention also provides a method for preparing the low-temperature neutral protease (Chprotease), specifically: inserting the low-temperature neutral protease gene fragment shown in SEQ ID NO.1 into the expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-Chprotease; transforming the recombinant plasmid pPIC9K-Chprotease into host cells; screening for positive recombinants and inducing expression; centrifuging to remove the supernatant after expression is complete, thus obtaining the product.

[0015] Furthermore, the host cell is Pichia pastoris GS115.

[0016] Furthermore, the method for preparing the low-temperature neutral protease includes the following steps:

[0017] Step S1. Extract total RNA from Colwellia holmium culture, reverse transcribe it into the first strand of cDNA, use the first strand of cDNA as a template, amplify the low-temperature neutral protease gene by PCR, purify it, and obtain the gene fragment of the protease Chprotease.

[0018] Step S2. The gene fragment of the protease Chprotease obtained in step S1 is ligated to the expression vector pPIC9K to obtain the recombinant plasmid pPIC9K-Chprotease;

[0019] Step S3. The recombinant plasmid pPIC9K-Chprotease obtained in step S2 was linearized with the enzyme Sal I and then electroporated into Pichia pastoris cells. Positive clones were screened to obtain recombinant Pichia pastoris cells GS115-pPIC9K-Chprotease, which was then induced to express. After expression, the supernatant was removed by centrifugation to obtain the final product.

[0020] Furthermore, the primers for PCR amplification in step S1 are shown in SEQ ID NO.3 to SEQ ID NO.4.

[0021] Furthermore, the treatment conditions for inducing expression in step S3 are as follows: the amount of methanol added is 0.5-1% of the liquid culture medium, the induction time is 4 days, and the pH of the induction culture medium is 6.5.

[0022] In addition, the present invention also provides the application of the aforementioned low-temperature neutral protease in the hydrolysis of proteins.

[0023] The *Colwellia hornerae* strain provided in this invention was purchased from Ningbo Testobio Biotechnology Co., Ltd., strain number: TS914050, brand: Testobio, strain number: CIP 105821, WDCM culture preservation number 759. The plasmid pPIC9K was purchased from Thermo Fisher Scientific.

[0024] Fish protein hydrolysates (FPH) are protein hydrolysates obtained through the hydrolysis of fish protein. Enzymatic hydrolysates, in particular, are widely used in fish protein hydrolysis due to their advantages of mild conditions, ease of control, high specificity, and ability to target specific proteins. Fish protein hydrolysates not only retain the high nutritional value of fish protein but also possess unique physiological properties and biological activities. Studies have found that fish protein hydrolysates have various physiological functions, including antioxidant properties, blood pressure reduction, regulation of lipid metabolism, and immune enhancement. However, factors such as the source of the protease, hydrolysis time, temperature, and pH value can significantly affect the bioactive products of the hydrolysates.

[0025] This invention provides a low-temperature neutral protease, Chprotease, derived from *Colwellia hornerae*. This Chprotease exhibits high enzyme activity within the temperature range of 15°C to 35°C, maintaining above 60% at 15°C, and high activity within the pH range of 6.5 to 7.5. It is a neutral low-temperature metalloproteinase that can maintain high activity at low or room temperature. This recombinant low-temperature neutral protease, Chprotease, can be used in the hydrolysis of fish protein. Specifically, using fish meal as a substrate at a concentration of 3.0%, and under the catalysis of 100 U / g Chprotease, the reaction is carried out at 25°C and pH 7.0 for 100 min. The fish meal hydrolysate exhibits good antioxidant properties, with a DPPH free radical scavenging rate greater than 60%. Furthermore, the fish meal hydrolysate also possesses good antibacterial activity, which can further broaden the application of the antioxidant and antibacterial activities of fish protein hydrolysates.

[0026] Compared with existing technologies, the low-temperature neutral protease Chprotease provided by this invention has the following advantages:

[0027] (1) This invention provides a new gene resource that can express low-temperature neutral protease, and combines the gene with a plasmid to produce neutral protease in a strain, thereby enriching the source of neutral protease.

[0028] (2) The low-temperature neutral protease Chprotease derived from Colwellia hornerae provided by this invention has high enzyme activity in the range of 15℃ to 35℃, and still maintains more than 60% at 15℃. It also has high activity in the range of pH 6.5 to 7.5. It can maintain high activity at low temperature or room temperature. It can not only simplify the hydrolysis steps of fish protein, but also effectively improve the hydrolysis ability of fish protein, thereby achieving the effect of reducing costs.

[0029] (3) The low-temperature neutral protease Chprotease derived from Colwellia hornerae provided by this invention has the advantages of mild reaction conditions, low energy consumption and environmental friendliness, and has broad application prospects, which has broadened the way for its industrial application. Attached image description:

[0030] Figure 1 This is a schematic diagram of the recombinant pPIC9K-Chprotease expression vector.

[0031] Figure 2 A schematic diagram of the recombinant Pichia pastoris expression strain for PCR verification.

[0032] Figure 3 The figure shows the temperature tolerance results of the recombinant protease Chprotease.

[0033] Figure 4 This is a graph showing the pH tolerance results of the recombinant protease Chprotease.

[0034] Figure 5 The graph shows the stability results of the recombinant protease Chprotease at different temperatures.

[0035] Figure 6 The figure shows the effect of different metal ion treatments on the activity of the recombinant protease Chprotease.

[0036] Figure 7 The graph shows the change in the antioxidant scavenging rate of fish meal protein peptides over time. Detailed Implementation

[0037] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic ideas of the invention, but as long as they do not depart from the basic ideas of the invention, they are all within the scope of the invention. Unless otherwise specified, the experimental methods used in the experiments involved in this invention are conventional methods; unless otherwise specified, the materials and reagents used in the examples of this invention are commercially available biochemical experimental materials.

[0038] Example 1: Construction of Pichia pastoris engineered strain containing the low-temperature neutral protease Chprotease

[0039] The structural diagram of the recombinant expression vector pPIC9K-Chprotease is shown below. Figure 1 As shown. The recombinant vector was transformed into Pichia pastoris GS115, and positive recombinants were screened. The specific operation steps are as follows:

[0040] Step S1. Extract total RNA from Colwellia holmium culture and reverse transcribe it into the first strand of cDNA. Using the first strand of cDNA as a template, design upstream and downstream primers F-Chp and R-Chp based on the gene sequence of the predicted protease in the Colwellia holmium genome in the NCBI database for PCR amplification. Detect and purify the PCR amplification product by 1% agarose gel electrophoresis and recover the gene fragment of the protease Chprotease from Colwellia holmium using a PCR recovery kit.

[0041] The sequences of the upstream and downstream primers F-Rha and R-Rha are shown in Table 1:

[0042] Table 1 Primers for PCR amplification

[0043]

[0044] The PCR amplification conditions were as follows: pre-denaturation at 98℃ for 4 min; high-temperature denaturation at 98℃ for 10 s, annealing at 55℃ for 15 s, extension at 72℃ for 2 min, for 30 cycles; and final denaturation at 72℃ for 10 min.

[0045] Step S2. The purified Chprotease gene fragment and expression vector pPIC9K from *Colletotrichum repens* obtained in Step S1 were digested with enzymes. The Chprotease gene fragment and pPIC9K DNA fragment were recovered and mixed at a volume ratio of 3:1. The mixture was ligated overnight at 16°C using T4 ligase. The ligation product was transformed into *E. coli* TOP10 competent cells. The transformation product was plated on LB agar plates containing 100 mg / L ampicillin and incubated overnight at 37°C. Single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium containing 100 mg / L ampicillin. The mixture was incubated overnight at 37°C with shaking. The plasmid was extracted and identified by enzyme digestion. The sequence of the plasmid after enzyme digestion was determined. The Chprotease coding gene was 1524 bp in size, and the nucleotide sequence was shown in SEQ ID NO.1, yielding the recombinant plasmid pPIC9K-Chprotease.

[0046] Step S3. The recombinant plasmid pPIC9K-Chprotease obtained in step S2 was linearized with the enzyme Sal I and then electroporated into Pichia pastoris cells. Positive clones were screened to obtain recombinant Pichia pastoris cells GS115-pPIC9K-Chprotease. Then, induction expression was performed. The induction expression conditions were as follows: the amount of methanol added was 0.5% of the liquid culture medium, the induction time was 4 days, the pH of the induction medium was 6.5, and the supernatant was removed by centrifugation after expression was completed.

[0047] (1) PCR was used to verify recombinant Pichia pastoris cells GS115-pPIC9K-Chprotease. The verification results are as follows: Figure 2 As shown in the figure. Lane M represents standard marker DNA of different molecular weights, and lanes 1-3 represent PCR bands (3 replicates) of three recombinant Pichia pastoris strains containing the pPIC9K-Chprotease plasmid. The results indicate that the Chprotease low-temperature neutral protease gene has been successfully recombined into Pichia pastoris.

[0048] (2) The nucleotide sequence (SEQ ID NO.1) of the low-temperature neutral protease Chprotease is shown below:

[0049]

[0050] (3) The amino acid sequence (SEQ ID NO.2) of the low-temperature neutral protease Chprotease is shown below:

[0051] MKMNLTKLSLATLLALSASTSVMASNGVNANHSNMNGKSINAVTQLSADYSFKAGKSFSTVKGNSKQKAQMFYKGVPVFGQSLVLEQDNAGRNVGTQGSLMVNISDDLGSVTPGMLRGKALGKLKQL LGHNNIKNSKTELVIYVNSANKAQLAYRVEYLASDSEVPSRPMAFIDANNGDVLLSWQGINHAKSGKGKPGSGGGGGSTTPTTLTGPGGNAKTGIYYYGTDFANIIATSDGSTCTLDSPNVITTDMG NSTRRGSTAQVACGDTGQDLTNGAFAPMNDAQAFGNVIFDMYSDWYGVTPLSQKLEMRVHYGRNYENAFWDGTAMSFGDGATTFHPLVSLDVSAHEVSHGVTEQRSGLVYSGESGGMNEAFSDMAGE AAENYMHGSNDWMVGEQIFKGNGALRYMDDPTKDGRSIAHASDMTSGLDVHLSSGVYNKAFYLLATTNGWTVQDAFGVMLRANNLYWTPSSTFDAGACGVESAATDLGLNSSDVTAAFATVGVNCP*.

[0052] Example 2: Enzyme activity determination and activity analysis of recombinant low-temperature neutral protease Chprotease

[0053] (1) Preparation of recombinant low-temperature neutral protease Chprotease:

[0054] The Pichia pastoris expression strain carrying pPIC9K-Chprotease obtained in Example 1 was inoculated into YPD medium and cultured at 28°C for 1 day. It was then transferred to BMGY medium at 1% (v / v). After 1 day of culture, the centrifuged cells were re-inoculated into fresh BMGY medium. During the culture period, 1% (v / v) methanol was added daily to induce keratinase expression. After 3–5 days of continued culture, the supernatant was collected by centrifugation to obtain the crude enzyme solution.

[0055] (2) Enzyme activity assay method:

[0056] Enzyme activity is defined as the number of micromoles of tyrosine divided by the number of minutes of time, yielding a measured unit value of protease activity. This is achieved by introducing the corresponding volume value to obtain the enzyme activity value in units per mL. To measure the activity of a solid protease sample diluted in enzyme dilution buffer, the unit / mL enzyme activity value can be divided by the original solid concentration used in the assay, expressed in mg / mL, to obtain the enzyme activity value per unit / mg.

[0057] Take 100 μL of crude enzyme solution and add 100 μL of 0.65% casein solution to each sample bottle, and allow them to stabilize in a 25°C water bath for about 5 minutes. Then, add different volumes of the enzyme solution to the three test sample bottles excluding the blank control bottle; mix by vortex and incubate at 25°C for 10 minutes to observe the protease activity and subsequent tyrosine release during this incubation period. After 10 minutes of incubation, add 100 μL of TCA reagent to each tube to terminate the reaction. Then, add an appropriate amount of enzyme solution to each tube, including the blank tube, so that the final volume of enzyme solution in each tube is 300 μL. This is to meet the requirements of the enzyme's absorbance value and to ensure that the final volume of each tube is equal. Incubate the solution at 37°C for 30 minutes. Then, measure the protease activity in the crude enzyme solution at 25°C; it is 22.82 U / mL.

[0058] (3) Determine the optimal pH for enzyme reaction:

[0059] Buffer solutions with pH values ​​of 5.0–10.0 were prepared using a citrate-sodium citrate buffer system (pH 4.0–6.0), phosphate-sodium phosphate buffer (pH 6.0–8.0), Tris-HCl buffer (pH 8.0–9.0), and glycine-NaOH. 100 μL of 13% casein solution was added to each sample vial, along with 100 μL of buffer solutions at different pH values. These were then stabilized in a 25°C water bath for approximately 5 minutes. Next, 100 μL of crude enzyme solution was added to each of the different pH sample vials. The mixture was vortexed and incubated at 25°C for 10 minutes. After 10 minutes, 100 μL of TCA reagent was added to each vial to terminate the reaction. Then, an appropriate amount of enzyme solution was added to each vial, including the blank vial, to ensure a final enzyme solution volume of 300 μL per vial. The absorbance was then measured using a microplate reader at a wavelength of 405 nm. Each group was tested in triplicate. Assuming the highest enzyme activity is 100%, plot a line graph showing the effect of pH on enzyme activity.

[0060] Enzyme activity was measured at 25°C under different pH conditions (5.0–10.0, 100 mmol / L sodium phosphate buffer). The optimal reaction pH for the recombinant low-temperature neutral protease Chprotease was found to be 7.0. The results are as follows: Figure 4 .

[0061] (4) Determine the temperature stability of the enzyme:

[0062] Prepare a sodium phosphate buffer solution with a pH of 7.0. Add 100 μL of 0.65% casein solution to each sample vial and allow them to stabilize in a 25°C water bath for approximately 5 minutes. Then, add 100 μL of crude enzyme solution to each sample vial. Mix by rotation and incubate at 10, 15, 20, 25, 30, 35, and 40°C for 10 minutes. After 10 minutes, add 100 μL of TCA reagent to each vial to terminate the reaction. Then, add an appropriate amount of enzyme solution to each vial, including the blank vial, so that the final volume of enzyme solution in each vial is 300 μL. Measure the absorbance at a wavelength of 405 nm using a microplate reader. Each group is divided into three replicates. Assuming the highest enzyme activity is 100%, plot a line graph of the effect of temperature on enzyme activity. It was found that the recombinant low-temperature neutral protease Chprotease is more stable under low-temperature conditions. After 10 minutes at 15°C, the activity still retains more than 60%, as shown in the results. Figure 5 .

[0063] (5) The effect of metal ions on protease activity:

[0064] Prepare a sodium phosphate buffer solution with a pH of 7.0. Add 100 μL of 0.65% casein solution to each sample vial and allow them to stabilize in a 25°C water bath for approximately 5 minutes. Take 10 μL of a 100 mmol / L sodium phosphate buffer solution. + K + Ca 2+ Mg 2+ Zn 2+ Cu 2+ Co 2+ Ten metal ions and the metalloproteinase inhibitor EDTA (pH 7.0) were mixed with 90 μL of crude enzyme solution to make the final ion concentration 10 mmol / L. Except for the ion concentration, the other conditions of the protease activity assay were the same as those in (2) above. The enzyme activity measured under the conditions of (2) was taken as 100%. The effect of each treatment on enzyme activity was detected. The experiment was repeated three times, and the significance of differences was analyzed using Sigmaplot software. The results are as follows. Figure 6 .

[0065] Example 3: Hydrolysis of fishmeal by recombinant low-temperature neutral protease Chprotease

[0066] Fish meal was used as the reaction substrate, and the reaction was carried out at 25°C and pH 7.0 for 1–3 h under the catalysis of the protease Chprotease. The antioxidant effect of hydrolyzed fish meal was monitored.

[0067] (1) Preparation of defatted fish meal:

[0068] 30g of fishmeal was placed in a 100mL Erlenmeyer flask. Using 90% ethanol as the solvent, 10% ethyl acetate was added, resulting in a total solvent-to-solid ratio of 10:1 (v / w) ml / g. The mixture was soaked for at least 3 hours. The flask was then placed in a constant-temperature water bath, equipped with a stirrer and reflux device, and extracted at 70.0–75.0℃ for 45 minutes each time. The slurry was then filtered, and the filter cake was washed two to three times with 20mL of 95% ethanol. After washing, the filter cake was transferred back to the flask, and a certain volume of 90% ethanol solution was added to continue the detoxification and deoiling process, repeating this process six times. After multiple treatments, the filter cake was placed in a vacuum drying oven and dried under reduced pressure at 60℃ to remove the solvent. It was then pulverized and passed through an 80-mesh sieve, and finally freeze-dried to obtain a crude sample of crispy tilapia protein.

[0069] (2) Effect of hydrolysis time on the hydrolysis effect of defatted fishmeal:

[0070] Under the conditions of 100 U / g enzyme dosage, 3.0% substrate concentration, 25℃ hydrolysis temperature, and pH 7.0, the hydrolysis times were set to 20 min, 60 min, 100 min, 140 min, and 180 min. The optimal hydrolysis time was selected by comparing the antioxidant scavenging rate of fishmeal protein hydrolysates.

[0071] (3) Determination of DPPH free radical scavenging ability:

[0072] Take 1 mL of sample solution, add 0.15 mol / L DPPH solution (dissolved in 95% ethanol), mix well, and react in the dark at room temperature for 30 min. Measure the absorbance (A) at a wavelength of 517 nm. i The blank group consisted of 3 mL of 95% ethanol solution instead of DPPH solution, with 1 mL of sample solution added and mixed. The absorbance was measured at 517 nm. j The control group consisted of 3 mL of DPPH solution with 1 mL of 95% ethanol, and its absorbance (A0) was measured at 517 nm. The formula for calculating the inhibition rate R of the product against DPPH is as follows:

[0073] R(%)=[1—(Ai—A j ) / A0]×100%

[0074] (4) Experimental results:

[0075] While maintaining an enzyme dosage of 100 U / g, a substrate concentration of 3.0%, a pH of 7.0, and a hydrolysis temperature of 25℃, the changes in the antioxidant scavenging rate of fishmeal protein peptides over time were as follows: Figure 7 As shown. By Figure 7It can be seen that when the hydrolysis time is less than 100 min, the antioxidant scavenging rate of fish meal protein peptides decreases; when the hydrolysis time is more than 100 min, the antioxidant scavenging rate of fish meal protein peptides does not change significantly compared with that at 120 min. Therefore, a hydrolysis time of 100 min is selected as the optimal hydrolysis time.

[0076] Example 4: Antibacterial test of tilapia protein hydrolysate

[0077] 1. Test materials:

[0078] The fishmeal hydrolysate prepared in Example 3 was tested with the following bacterial strains (commercially available): Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Vibrio cholerae CICC 23794, and Listeria innocua CICC 10297.

[0079] 2. Test methods:

[0080] The antibacterial effect was determined by referring to the paper disc diffusion method in Han Wenyu's article "Detection Techniques for Pathogenic Bacteria" and making slight modifications.

[0081] The tested strains were prepared into 0.3 × 10⁻⁶ saturated solutions. 8 A bacterial suspension of CFU / mL was evenly spread at a rate of 0.1 mL / cm² on nutrient agar plates (9 cm in diameter). Sterile filter paper discs (approximately 8-10 mm in diameter) were then attached to each disc. 30 μL of fishmeal protein hydrolysate was added to each disc, while a blank control group was prepared by adding 30 μL of sterile physiological saline. The plates were incubated at 30°C for 12-18 hours, and the size of the inhibition zone was measured to determine the antimicrobial effect of the antimicrobial peptide. The enzymatic hydrolysate of fishmeal by Bacillus subtilis neutral protease served as a control group.

[0082] 3. Test Results:

[0083] The experimental results are shown in Table 2.

[0084] Table 2 Antibacterial test data of tilapia protein hydrolysates

[0085]

[0086] As shown in Table 2, the hydrolysate of fishmeal hydrolyzed by the low-temperature neutral protease Chprotease provided by this invention has good antibacterial activity, especially significantly improving the inhibitory effect on Escherichia coli ATCC 25922, Vibrio cholerae CICC 23794 and Listeria innocua CICC 10297.

[0087] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for hydrolyzing fishmeal, characterized in that, Includes the following steps: Step S1, Preparation of defatted fish meal: Take 30 g of crispy tilapia fish powder and place it in a 100 mL Erlenmeyer flask. Use 90% ethanol as solvent and add 10% ethyl acetate. The total solvent-to-material ratio is 10:1 ml / g. Soak for 3 h. Place the Erlenmeyer flask in a constant temperature water bath, equip it with a stirring and reflux device, and extract at 70.0~75.0℃ for 45 min each time. After extraction, filter the slurry. Wash the filter cake two to three times with 20 mL of 95% ethanol. After washing, transfer the filter cake back into the Erlenmeyer flask and add 90% ethanol solution to continue the detoxification and deoiling operation. Repeat 6 times. Place the filter cake in a vacuum drying oven and dry under reduced pressure at 60℃ to remove the solvent. Crush it through an 80 mesh sieve and freeze dry to obtain crude sample of crispy tilapia protein. Step S2, Hydrolysis treatment: Using the crude tilapia protein sample obtained in step S1 as a substrate, with a substrate concentration of 3.0%, the mixture was reacted for 100 min at 25°C and pH 7.0 under the catalysis of 100 U / g low-temperature neutral protease. The amino acid sequence of the low-temperature neutral protease is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1.

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