Trichoderma strain capable of expressing lactase and protease and use thereof
By screening and identifying Trichoderma strain CCTCC NO: M 20221323, the problem of insufficient stability and activity of lactase and protease in the existing technology has been solved, realizing its application advantages in industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NINGBO XINUOYA MARINE BIOTECH CO LTD
- Filing Date
- 2022-10-12
- Publication Date
- 2026-08-04
AI Technical Summary
The lack of highly stable and active Trichoderma strains containing lactase and protease in existing technologies limits their application in industrial production.
A Trichoderma sp. strain (CCTCC NO: M 20221323) was screened and identified. It expressed lactase and protease. The optimal reaction temperature for lactase was 60℃ and for protease was 50℃. Both strains showed good stability and activity under acidic conditions.
The lactase and protease expressed by this strain exhibit high activity and stability in industrial production, demonstrating significant application advantages.
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Figure CN116103159B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, specifically to a Trichoderma strain capable of expressing lactase and protease and its applications. Background Technology
[0002] Lactase, or β-galactoside galactohydrolase (EC 3.2.1.23), catalyzes the hydrolysis of lactose to produce galactose and glucose and possesses some transglycosylation activity. Lactase has wide applications in food, medicine, and environmental fields, primarily addressing lactose intolerance in food and medicine. Natural sources of lactase are abundant, widely distributed in animals, plants, and microorganisms; however, current research indicates that only microbially produced lactases have industrial applications. Among these, molds such as *Trichoderma* and *Aspergillus oryzae* have not shown any toxicity in safety tests and are considered safe strains.
[0003] Proteases are a class of enzymes with significant commercial applications. Based on their origin, they can be classified into animal proteases, plant proteases, and microbial proteases. Among these, microbial proteases have wide applications in the food and pharmaceutical industries. Microorganisms can produce proteases through fermentation using agricultural byproducts, resulting in low production costs and ease of artificial modification. These proteases exhibit broad substrate specificity, and due to the diverse living environments of microorganisms, their adaptability is also very wide. According to their optimal reaction pH, proteases can be classified into acidic, neutral, and alkaline types. Acidic proteases have been widely used in the food, pharmaceutical, and leather industries, demonstrating significant economic benefits in the production of alcoholic beverages and soy sauce, as well as in leather processing.
[0004] Therefore, screening out a Trichoderma strain that can express highly stable and highly active lactase and protease is of great significance for the industrial production of lactase and protease. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art: to provide a Trichoderma strain that can express highly stable and highly active lactase and protease and its applications.
[0006] The technical solution of the present invention is as follows: a Trichoderma strain capable of expressing lactase and protease, wherein the strain is a Trichoderma sp. strain, the deposit date is August 24, 2022, the depositary institution is China Center for Type Culture Collection (CCTCC), and the deposit number is CCTCC NO: M 20221323.
[0007] The nucleotide sequence of the Trichoderma strain capable of expressing lactase and protease is shown in SEQ ID NO: 1.
[0008] The present invention also improves the application of the aforementioned Trichoderma strain capable of expressing lactase and protease for expressing lactase and / or protease.
[0009] The optimal reaction temperature for lactase activity expressed by the Trichoderma strain is 60℃.
[0010] The lactase expressed by the Trichoderma strain exhibits good thermal stability at 20-50℃.
[0011] The optimal reaction pH for the lactase activity expressed by the Trichoderma strain is 4.
[0012] The optimal reaction temperature for the protease activity expressed by the Trichoderma strain is 50℃.
[0013] The protease expressed by the Trichoderma strain exhibits good thermal stability at 20-50℃.
[0014] The optimal reaction pH for the protease activity expressed by the Trichoderma strain is 2.
[0015] The beneficial effects of this invention are as follows: This invention isolates and identifies a Trichoderma strain, with accession number CCTCCNO: M 20221323, accession date August 24, 2022, and accession address Wuhan University, Wuhan. When this strain is used to express lactase and / or protease, the optimal reaction pH for both lactase and protease is acidic, exhibiting good stability and high activity, which has significant advantages in industrial production. Attached Figure Description
[0016] Figure 1 The colony morphology of the Trichoderma strain in Example 4 after 5 days of culture on PDA solid medium; the left image is the front view, and the right image is the back view;
[0017] Figure 2 This is a microscopic image of the Trichoderma strain in Example 4;
[0018] Figure 3 This is a phylogenetic tree constructed based on sequence results for the Trichoderma strain in Example 6;
[0019] Figure 4 This is a graph showing the effect of different temperatures on lactase activity in Example 7;
[0020] Figure 5 This is a graph showing the effect of different pH values on lactase activity in Example 8;
[0021] Figure 6 This is a graph showing the effect of different temperatures on protease activity in Example 9;
[0022] Figure 7 This is a graph showing the effect of different pH values on protease activity in Example 10. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.
[0024] Example 1: Screening and obtaining Trichoderma strains.
[0025] 1. Preparation of culture medium
[0026] PDA medium: Wash and peel potatoes, weigh 200g, cut into small pieces, boil in pure water for 30 minutes, filter through eight layers of gauze to remove residue, cool, and add water to a final volume of 1L, pH set to natural. If using a solid medium, add 20g of agar, dispense, and sterilize at 121℃ for 20 minutes. 50% glucose: Weigh 50g of glucose, dissolve in water and bring to a final volume of 100mL, sterilize at 115℃ for 30 minutes.
[0027] PDA solid plates: Cool the sterilized PDA medium to about 60°C, add 20 g / L glucose and 20 mg / mL X-gal, pour into plates, and let solidify before use.
[0028] 2. Screening and acquisition of strains
[0029] Sample collection: Seawater samples were collected on May 1, 2022, from the nearshore mudflats of Wenchang City, Hainan Province (E 110°49′23″, N19°32′12″) and stored in sterile bottles.
[0030] Strain screening and acquisition: After serial dilution with sterile water, the samples were distributed on PDA solid medium by plate spreading, numbered, and sealed with film. They were then incubated upside down in a 30℃ biochemical incubator for 3-5 days. Initial plate screening revealed a blue-colored fungus, which was then inoculated onto PDA solid medium for enrichment and purification.
[0031] Example 2: Determination of lactase activity in Trichoderma strains.
[0032] 1. Preparation of buffer solutions and reagents
[0033] Acetate buffer: Pipette 25 mL of 2N acetic acid, add approximately 300 mL of water, and adjust the pH to 4.5 with 2N NaOH solution. Transfer the solution to a 500 mL volumetric flask and dilute to volume. 10% Na₂CO₃ solution: Weigh 50 g of anhydrous sodium carbonate, dissolve in a small amount of deionized water, and dilute to volume in a 500 mL volumetric flask. Substrate: Weigh 185.0 mg of ONPG (o-nitrophenyl-β-D-galactopyranoside), dissolve in 40 mL of acetate buffer, and dilute to volume in a 50 mL volumetric flask. Prepare fresh before use, keep in a cool, dark place until use, and perform the assay within 2 hours. 2 mM o-nitrophenol: Weigh 139.0 mg of o-nitrophenol into a 500 mL volumetric flask, dissolve in 10 mL of 95% ethanol, dilute to the mark with 1% Na₂CO₃ solution, and mix thoroughly.
[0034] 2. Preparation of the o-nitrophenol standard curve
[0035] 2 mM o-nitrophenol was diluted to 0.10, 0.14, and 0.18 mM with 1% Na₂CO₃ solution. The absorbance of the three concentrations of o-nitrophenol standard solutions was measured in a 1 cm cuvette at 420 nm using a suitable spectrophotometer, and zeroing was performed with water. Linear regression analysis was performed on the absorbance values of each o-nitrophenol standard solution with the three concentrations (0.10, 0.14, and 0.18 mM).
[0036] 3. Obtaining crude lactase solution
[0037] Inoculate the Trichoderma on the PDA solid plate into the PDA liquid medium, culture at 30℃ and 150 r / min for 6 days with shaking, centrifuge at 4000 r / min and collect the supernatant, which is the crude enzyme solution.
[0038] 4. Lactase activity assay
[0039] Pipette 2 mL of substrate solution into a series of large test tubes and equilibrate in a 37°C water bath. At time zero, quickly add 0.5 mL of appropriately diluted enzyme solution to the equilibrated substrate, mix, and immediately return the test tubes to the water bath for reaction. After 15 minutes of reaction, add 2.5 mL of 10% sodium carbonate solution to all tubes, mix quickly, and remove from the water bath. Simultaneously, prepare a blank tube by adding 2 mL of substrate, 0.5 mL of deionized water, and 2.5 mL of 10% sodium carbonate solution. Add 20 mL of water to both the sample and blank tubes and mix thoroughly. Using a suitable spectrophotometer, measure the absorbance of each sample tube and the blank in a 1 cm cuvette at 420 nm, and zero the sample tube with water. Record the data and calculate. One unit of lactase (ALU) is defined as the amount of enzyme required to produce 1 μmol ONP per minute under these experimental conditions.
[0040] Example 3: Determination of protease activity in Trichoderma strains.
[0041] 1. Preparation of buffer solutions and reagents
[0042] 0.05M glycine-hydrochloric acid buffer: Dissolve 3.75g glycine in approximately 800mL of water. Add 1N hydrochloric acid to adjust the pH to 3.0, and bring the volume to 1000mL with water. TCA solution: Dissolve 18.0g trichloroacetic acid and 11.45g anhydrous sodium acetate in approximately 800mL of water. Add 21.0mL glacial acetic acid, and bring the volume to 1000mL with water. Substrate solution: Add 8mL 1N hydrochloric acid to approximately 500mL of water, and disperse 7.0g (anhydrous) casein into the solution with continuous stirring. Heat in a boiling water bath for 30 minutes, stirring occasionally, and cool to room temperature. Add 3.75g glycine and dissolve. Adjust the pH to 3.0 with 0.1N hydrochloric acid, and bring the volume to 1000mL with water.
[0043] 2. Preparation of Tyrosine Standard Curve
[0044] 181.2 mg of L-tyrosine, pre-dried to constant weight, was dissolved in 60 mL of 0.1 N hydrochloric acid, and the solution was diluted with water to 1000 mL. This solution contained 1.00 μmol of tyrosine per 1.0 mL. Diluted solutions were prepared from this stock solution to contain 0.10, 0.20, 0.30, 0.40, and 0.50 μmol / mL. Using water as a blank, the absorbance in a 1 cm cuvette at 275 nm was measured using a suitable spectrophotometer. A graph of absorbance versus tyrosine concentration (μmol / mL) was plotted.
[0045] 3. Protease activity assay
[0046] Add 10.0 mL of substrate solution to a series of large test tubes, stopper the tubes, and equilibrate in a 37°C water bath for 15 minutes. At time zero, add 2.0 mL of crude enzyme solution, appropriately diluted with glycine-hydrochloric acid buffer, to the equilibrated substrate. Replace the enzyme solution with 2 mL of glycine-hydrochloric acid buffer to create a substrate blank. After reacting for 30 minutes, add 10 mL of TCA solution to each test tube. Prepare enzyme blanks by adding 10 mL of substrate solution, 10 mL of TCA solution, and 2 mL of enzyme solution in that order. Heat all tubes in a water bath for 30 minutes to completely coagulate the precipitated protein. After cooling the tubes in an ice bath for 5 minutes, filter the solution; the filtrate must be very clear. Measure the absorbance in a 1 cm cuvette at 275 nm using a suitable spectrophotometer. Correct for errors by subtracting the absorbance of the corresponding enzyme blank.
[0047] Example 4: Morphological characteristics of Trichoderma strains.
[0048] The isolated and purified *Trichoderma* strain was transferred to PDA solid plates and incubated at 30°C for 5 days. Colony morphology was observed, colony diameter was measured, and colony shape and color were recorded. Morphology was observed under a microscope. After 5 days of incubation, the *Trichoderma* colony diameter was 8.5 cm. The mycelium grew rapidly, appearing as white, fluffy hairs, with similar color on both sides. See attached image for colony morphology. Figure 1 Under a microscope, the hyphae are long and septate; their microscopic morphology is shown in... Figure 2 .
[0049] Example 5: Effect of carbon source on the growth of Trichoderma strains.
[0050] Using starch, sucrose, and carboxymethyl cellulose as carbon sources, respectively, and replacing glucose in PDA solid medium in equal amounts, Trichoderma strains were transferred to plates and cultured at 30°C for 5 days. Trichoderma strains were able to grow on the medium and had similar morphology.
[0051] Example 6: Molecular biological identification of Trichoderma strains.
[0052] 1. Fungal DNA Genome Extraction
[0053] Take the culture medium from Example 2, centrifuge it, take a portion of the bacterial cells and place them in a mortar, add liquid nitrogen and grind them thoroughly. Collect the ground bacterial cells in a centrifuge tube and store them in a -20°C refrigerator for later use.
[0054] Take 200 mg of the homogenized bacterial cells ground with liquid nitrogen, add 3% CTAB, incubate at 65°C for 45 min, and centrifuge at 13000 rpm for 10 minutes. Take the supernatant and add an equal volume of phenol:chloroform:isoamyl alcohol (volume ratio 25:24:1), centrifuge at 13000 rpm for 10 minutes. Take the supernatant and add an equal volume of chloroform:isoamyl alcohol (volume ratio 24:1), centrifuge at 13000 rpm for 10 minutes. Add two volumes of pre-cooled anhydrous ethanol to the supernatant, incubate at -20°C for 20-30 minutes, and centrifuge at 13000 rpm for 10 minutes. Discard the supernatant, wash the precipitate with 200 μL of 70% ethanol, and centrifuge at 13000 rpm for 10 minutes. Discard the supernatant and dry the precipitate at room temperature. Add 30 μL of water to completely dissolve the DNA and store at -20°C.
[0055] 2. PCR amplification
[0056] The extracted bacterial DNA was processed using primer ITS1, whose sequence is shown in SEQ ID NO: 2.
[0057] PCR amplification was performed using primers (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS2 (5′-GCTGCGTTCTTCATCGATGC-3′) with the sequence shown in SEQ ID NO: 3. The PCR reaction system consisted of: 25 μL 2×TAQ enzyme, 2 μL ITS1 primer, 2 μL ITS2 primer, 5 μL template, and 16 μL sterile water. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 60 s, and 72℃ extension for 60 s, for 30 cycles; 72℃ extension for 7 min; and storage at 4℃.
[0058] 3. rDNA-ITS sequencing and analysis
[0059] The target PCR product was recovered and purified using a DNA gel rapid purification kit (Beijing TransGen Biotech Co., Ltd.). The purified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The gene sequence is shown in SEQ ID NO: 1.
[0060] The obtained sequences were aligned to NCBI using BLAST, analyzed using Mega 4.0 software, and a phylogenetic tree was constructed using the Neighbor-Joining method. Figure 3 As shown.
[0061] Phylogenetic analysis revealed that this strain is highly related to Trichoderma strains. Based on morphological characteristics, the strain was identified as belonging to the Trichoderma genus of the family Hypocreaceae.
[0062] The strain CY5104-1 was deposited at the China Center for Type Culture Collection (CCTCCNO: M 20221323) on August 24, 2022. The depositary address is Wuhan University, Wuhan.
[0063] Example 7: Effect of temperature on lactase activity
[0064] 1. Optimal reaction temperature of lactase
[0065] The crude enzyme solution was appropriately diluted and enzymatically hydrolyzed at reaction temperatures of 20, 30, 37, 43, 50, 60, 70, and 80°C, respectively. Lactase activity was determined according to the method described in Example 2. The enzyme activity measured at the optimal reaction temperature was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The relative enzyme activities are shown in the table below:
[0066] Relative enzyme activity / % 12.3 19.9 24.4 47.2 56.5 100 19.8 9.98
[0067] The relative enzyme activity change curve is as follows: Figure 4 As shown, the lactase expressed by Trichoderma exhibits the highest activity at a reaction temperature of 60°C.
[0068] 2. Thermostability of Lactase
[0069] The crude enzyme solution was appropriately diluted and incubated at 20, 30, 37, 43, 50, 60, 70, and 80°C for 30 minutes each, then immediately cooled in an ice bath. Lactase activity was measured under optimal reaction conditions. The enzyme activity measured at different temperatures without incubation was taken as 100%, and the relative enzyme activity at each temperature was calculated. The relative enzyme activities are shown in the table below:
[0070] Relative enzyme activity / % 85.6 90.4 100 96.6 97.6 69.9 20.4 16.4
[0071] The relative enzyme activity change curve is as follows: Figure 4 As shown, the lactase expressed by Trichoderma exhibits good thermostability within the temperature range of 20-50℃, with relative enzyme activities all exceeding 85%.
[0072] Example 8: Effect of pH on lactase activity
[0073] 1. Optimal pH for lactase reaction
[0074] The crude enzyme solution was appropriately diluted and enzymatically hydrolyzed at the optimal reaction temperature and at pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0, respectively. Lactase activity was determined according to the method described in Example 2. The enzyme activity measured at the optimal reaction pH was taken as 100%, and the relative enzyme activity at different pH values was calculated. The relative enzyme activities are shown in the table below:
[0075] Relative enzyme activity / % 72.0 100 89.8 51.0 29.2 23.8 23.5 24.2
[0076] The relative enzyme activity change curve is as follows: Figure 5 As shown, the lactase expressed by Trichoderma exhibits the highest activity at pH 4, and its activity remains above 70% at pH 3-5.
[0077] Example 9: Effect of temperature on protease activity
[0078] 1. Optimal reaction temperature of proteases
[0079] The crude enzyme solution was appropriately diluted and enzymatically hydrolyzed at reaction temperatures of 20, 30, 37, 43, 50, 60, 70, and 80°C, respectively. The protease activity was determined according to the method described in Example 3. The enzyme activity measured at the optimal reaction temperature was taken as 100%, and the relative enzyme activity at different temperatures was calculated. The relative enzyme activities are shown in the table below:
[0080] Relative enzyme activity / % 35.7 41.3 82.5 91.3 100 93.8 47.5 0
[0081] The relative enzyme activity change curve is as follows: Figure 6 As shown, the protease expressed by Trichoderma exhibits the highest activity at a reaction temperature of 50℃, and its activity remains above 80% in the range of 37-60℃.
[0082] 2. Thermal stability of proteases
[0083] The crude enzyme solution was appropriately diluted and incubated at 20, 30, 37, 43, 50, 60, 70, and 80°C for 30 minutes each, then immediately cooled in an ice bath. The protease activity was then measured under optimal reaction conditions. The enzyme activity measured at different temperatures without incubation was taken as 100%, and the relative enzyme activity at each temperature was calculated. The relative enzyme activities are shown in the table below:
[0084] Relative enzyme activity / % 72.5 100 85.5 87.0 82.6 56.6 40.8 0
[0085] The relative enzyme activity change curve is as follows: Figure 6 As shown, the proteases expressed by Trichoderma exhibit good thermostability within the temperature range of 20-50℃, with relative enzyme activities all exceeding 70%.
[0086] Example 10: Effect of pH on protease activity
[0087] 1. Optimal pH for protease reaction
[0088] The crude enzyme solution was appropriately diluted and enzymatically hydrolyzed at the optimal reaction temperature and at pH values of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The protease activity was determined according to the method in Example 3. The enzyme activity measured at the optimal reaction pH was taken as 100%, and the relative enzyme activity at different pH values was calculated. The relative enzyme activities are shown in the table below:
[0089] Relative enzyme activity / % 0 100 87.0 85.8 41.7 45.6 31.3
[0090] The relative enzyme activity change curve is as follows: Figure 7 As shown, the protease expressed by *Trichoderma* exhibits the highest activity at a reaction pH of 2, and its activity exceeds 85% at pH 2-4.
[0091] The above are merely exemplary embodiments of the present invention and do not constitute any limitation on the scope of protection of the present invention. All technical solutions formed by equivalent exchange or substitution fall within the scope of protection of the present invention.
Claims
1. A Trichoderma strain that can express lactase and protease, characterized in that: The strain is a Trichoderma sp. strain, deposited on August 24, 2022, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20221323.
2. Use of a Trichoderma strain expressing lactase and protease according to claim 1, characterized in that, Used to express lactase and / or protease.
3. Use of a Trichoderma strain expressing lactase and protease according to claim 2, characterized in that, The optimal reaction temperature for lactase activity expressed by the Trichoderma strain is 60℃.
4. Use of a Trichoderma strain expressing lactase and protease according to claim 3, characterized in that, The lactase expressed by the Trichoderma strain exhibits good thermal stability at 20-50℃.
5. Use of a Trichoderma strain expressing lactase and protease according to claim 4, characterized in that, The optimal reaction pH for the lactase activity expressed by the Trichoderma strain is 4.
6. Use of a Trichoderma strain expressing lactase and protease according to claim 5, characterized in that, The optimal reaction temperature for the protease activity expressed by the Trichoderma strain is 50℃.
7. Use of a Trichoderma strain expressing lactase and protease according to claim 6, characterized in that, The protease expressed by the Trichoderma strain exhibits good thermal stability at 20-50℃.
8. Use of a Trichoderma strain expressing lactase and protease according to claim 7, characterized in that, The optimal reaction pH for the protease activity expressed by the Trichoderma strain is 2.