CN116103159A

CN116103159A - Trichoderma strain capable of expressing lactase and protease and application thereof

Info

Publication number: CN116103159A
Application number: CN202211246375.7A
Authority: CN
Inventors: 林新月; 丁慧; 曹阳; 柳燕; 田�健; 诸辉
Original assignee: Ningbo Xinuoya Marine Biotechnology Co ltd
Current assignee: Ningbo Xinuoya Marine Biotechnology Co ltd
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2023-05-12

Abstract

The invention belongs to the field of microorganisms, and discloses a Trichoderma strain capable of expressing lactase and protease, which is Trichoderma (Trichoderma sp.) strain, the preservation date is 2022, 8 months and 24 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m20221323, the nucleotide sequence of the trichoderma strain capable of expressing lactase and protease is shown in SEQ ID NO:1, when the strain is applied to the expression of lactase and/or protease, the optimal reaction pH values of the lactase and the protease are acidic, so that the strain has good stability and high activity.

Description

Trichoderma strain capable of expressing lactase and protease and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to a trichoderma strain capable of expressing lactase and protease and application thereof.

Background

Lactase, beta-galactoside galactose hydrolase (beta-galactoside galactohydrolase, e.c. 3.2.1.23), has the ability to catalyze the hydrolysis of lactose to galactose and glucose and has some transglycosylation activity. Lactase has wide application in the fields of food, medicine, environment and the like, and is mainly used for solving lactose intolerance in the food and the medicine. Lactase is a natural source which is quite abundant and widely distributed in animals, plants and microorganisms, but the research at present finds that only lactase produced by microorganisms has industrial application value. Among them, the mold such as trichoderma and aspergillus oryzae is considered as a safe strain, since the mold does not have poisoning phenomenon in the safety test.

Proteases are a class of enzyme preparations with important commercial application value, and can be classified into animal proteases, plant proteases and microbial proteases according to the source. Among them, microbial protease has been widely used in the fields of foods, medicines, etc. The microorganism can ferment and produce protease by taking agricultural and sideline products as raw materials, the production cost is low, and the artificial transformation is easy to carry out. The protease has wide substrate specificity, and the adaptability of the enzyme is very wide due to the diversity of the living environments of microorganisms. The protease can be classified into acidity, neutrality and alkalinity according to the pH optimum of the reaction, wherein the acid protease is widely applied to industries such as food, medicine and leather, and has obvious economic effects in the production fields such as wine, soy sauce brewing and the like and leather treatment and the like.

Therefore, the screening of the trichoderma strain capable of expressing the lactase and the protease with high stability and high activity has important significance for industrial production of the lactase and the protease.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects in the prior art: provides a trichoderma strain capable of expressing lactase and protease with high stability and high activity and application thereof.

The technical scheme of the invention is as follows: a Trichoderma strain capable of expressing lactase and protease, which is Trichoderma (Trichoderma sp.) strain, the preservation date is 2022, 8 months and 24 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m20221323.

The nucleotide sequence of the trichoderma strain capable of expressing lactase and protease is shown in SEQ ID NO: 1.

The invention also provides application of the trichoderma strain capable of expressing lactase and protease, which is used for expressing lactase and/or protease.

The optimal reaction temperature of lactase enzyme activity expressed by the trichoderma strain is 60 ℃.

Lactase expressed by the trichoderma strain has good heat stability at 20-50 ℃.

The optimal reaction pH of lactase enzyme activity expressed by the trichoderma strain is 4.

The optimal reaction temperature of the protease activity expressed by the trichoderma strain is 50 ℃.

The protease expressed by the trichoderma strain has good thermal stability at 20-50 ℃.

The optimal reaction pH of the protease activity expressed by the trichoderma strain is 2.

The beneficial effects of the invention are as follows: the invention separates and identifies a trichoderma strain with the preservation number of CCTCC NO: m20221323, the preservation date is 2022, 8 and 24 days, and the preservation unit address is Wuhan university. When the strain is applied to expressing lactase and/or protease, the optimal reaction pH of the lactase and the protease is acidic, the stability is good, the activity is high, and the strain has great advantages in industrial production.

Drawings

FIG. 1 shows the colony morphology of the Trichoderma strain of example 4 when cultured on PDA solid medium for 5 d; the left graph is the front, and the right graph is the back;

FIG. 2 is a morphological diagram of Trichoderma strain of example 4 under a microscope;

FIG. 3 is a phylogenetic tree constructed based on the sequence results for Trichoderma strains of example 6;

FIG. 4 is a graph showing the effect of different temperatures on lactase enzyme activity in example 7;

FIG. 5 is a graph showing the effect of different pH values on lactase enzyme activity in example 8;

FIG. 6 is a graph showing the effect of different temperatures on protease activity in example 9;

FIG. 7 is a graph showing the effect of different pH values on protease activity in example 10.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples, but the present invention is not limited to the following specific examples.

Example 1: screening and obtaining the trichoderma strains.

1. Preparation of culture Medium

PDA medium: cleaning potato, peeling, weighing 200g, cutting into small pieces, boiling in pure water for 30 min, filtering with eight layers of gauze to remove residues, cooling, adding water to 1L, and naturally adjusting pH. If the culture medium is solid, adding agar 20g, packaging, and sterilizing at 121deg.C for 20 min. 50% glucose: 50g of glucose was weighed, dissolved in water and fixed to 100mL and sterilized at 115℃for 30 minutes.

PDA solid plate: the sterilized PDA culture medium is cooled to about 60 ℃, 20g/L glucose and 20mg/mL X-gal are added, the plate is poured, and the plate is solidified for standby.

2. Screening and obtaining of strains

Sample collection: seawater samples collected on offshore beach (E110 DEG 49 '23', N19 DEG 32 '12') from Wenchang, hainan province, 5 months 1, 2022 were stored in sterile bottles.

Strain screening and obtaining: after the sample is diluted by sterile water in a gradient way, the sample is distributed on a PDA solid culture medium in a flat plate coating way, the sample is numbered and a sealing film is attached, and the sample is inversely cultured for 3-5 days in a biochemical incubator at 30 ℃. Through preliminary screening, a strain of blue fungus is found on the plate, and the strain is inoculated on a PDA solid culture medium for enrichment and purification.

Example 2: lactase enzyme activity assay of Trichoderma strain.

1. Buffer solution and reagent preparation

Acetate buffer: 25mL of 2N acetic acid was taken up, about 300mL of water was added, and the pH was adjusted to 4.5 with 2N NaOH solution. The solution was transferred to a 500mL volumetric flask and the volume was fixed. 10% Na ₂ CO ₃ Solution: 50g of anhydrous sodium carbonate was weighed, dissolved with a small amount of deionized water, and the volume was fixed in a 500mL volumetric flask. A substrate: 185.0mg of ONPG (o-nitrophenyl-beta-D-galactose) was weighed, dissolved in 40mL of acetate buffer and fixed in a 50mL volumetric flask, and ready to use, kept in a cool and light place until use, and tested within 2 hours. 2mM o-nitrophenol: 139.0mg of o-nitrophenol was weighed into a 500mL volumetric flask, dissolved in 10mL of 95% ethanol, and treated with 1% Na ₂ CO ₃ The solution was diluted to scale and mixed thoroughly.

2. O-nitrophenol standard curve preparation

With 1% Na ₂ CO ₃ Solution 2mM o-nitrophenol was diluted to 0.10,0.14 and 0.18mM respectively, absorbance of three concentrations of o-nitrophenol standard solution was measured in a 1cm cuvette at 420nm using a suitable spectrophotometer, and zeroing was performed with water. Linear regression analysis was performed on absorbance values of each o-nitrophenol by standard solution concentrations (0.10,0.14 and 0.18 mM) of three o-nitrophenols.

3. Obtaining lactase crude enzyme liquid

Inoculating Trichoderma on a PDA solid plate into a PDA liquid culture medium, performing shake culture at 30 ℃ and 150r/min for 6 days, centrifuging at 4000r/min, and collecting supernatant to obtain crude enzyme liquid.

4. Lactase Activity assay

2mL of substrate solution was pipetted into a series of large test tubes and equilibrated in a 37℃water bath. At zero time, 0.5mL of the enzyme solution after proper dilution is quickly sucked up and added into the substrate with balanced temperature, and the mixture is mixed, and then the test tube is immediately placed into a water bath for reaction. After 15 minutes of reaction, 2.5mL of 10% sodium carbonate solution was added to all tubes, quickly mixed, and removed from the water bath. A blank tube was prepared and 2mL of substrate, 0.5mL of deionized water and 2.5mL of 10% sodium carbonate solution were added. To the sample and blank tube, 20mL of water was added and then thoroughly mixed. The absorbance of each tube and blank of the sample was measured using a suitable spectrophotometer in a 1cm cuvette at 420nm and zeroed with water. The data is recorded and calculated. One lactase unit (ALU) is defined as the amount of enzyme required to produce 1. Mu. Mol ONP per minute under the conditions of this assay.

Example 3: protease enzyme activity assay of Trichoderma strain.

1. Buffer solution and reagent preparation

0.05M glycine-HCl buffer: 3.75g glycine was dissolved in about 800mL water. 1N hydrochloric acid was added to the solution to pH 3.0 and the volume was set to 1000mL with water. TCA solution: 18.0g of trichloroacetic acid and 11.45g of anhydrous sodium acetate were dissolved in about 800mL of water, 21.0mL of glacial acetic acid was added, and the volume was fixed with water to 1000mL. Substrate solution: 8mL of 1N hydrochloric acid was added to about 500mL of water, and 7.0g (anhydrous base) casein was dispersed into the solution under continuous stirring. Heated in a boiling water bath for 30 minutes with occasional stirring and cooled to room temperature. 3.75g glycine was added and dissolved, adjusted to pH 3.0 with 0.1N hydrochloric acid, and the volume was set to 1000mL with water.

2. Preparation of tyrosine standard curve

181.2mg of L-tyrosine, which had been dried to a constant weight beforehand, was dissolved in 60mL of 0.1N hydrochloric acid, and the solution was diluted with water to 1000mL. The solution contained 1.00. Mu. Mol tyrosine per 1.0 mL. A dilution was prepared from this stock solution to contain 0.10,0.20,0.30,0.40 and 0.50. Mu. Mol/mL. The absorbance in a 1cm cuvette at 275nm was measured with a suitable spectrophotometer using water as a blank. The absorbance is plotted against tyrosine concentration (μmol/mL).

3. Protease Activity assay

10.0mL of substrate solution was added to a series of large test tubes, the tubes were stoppered, and equilibrated in a 37℃water bath for 15 minutes. At time zero, 2.0mL of crude enzyme solution, suitably diluted with glycine-HCl buffer, was added to the equilibrated substrate. 2mL glycine-hydrochloric acid buffer was added to the substrate as a substrate blank instead of the enzyme solution. After 30 minutes of reaction, 10mL of TCA solution was added to each tube. Enzyme blanks were prepared in the order of 10mL substrate solution, 10mL TCA solution and 2mL enzyme solution. All tubes were heated in a water bath for 30 minutes to allow the precipitated protein to solidify completely. After cooling the tube in an ice bath for 5 minutes, the filtrate must be very clear. Absorbance was measured in a 1cm cuvette at 275nm using a suitable spectrophotometer. Corrected by subtracting the absorbance of the corresponding enzyme blank.

Example 4: morphological characteristics of the Trichoderma strain.

Transferring the separated and purified trichoderma strain to a PDA solid plate, culturing at 30 ℃ for 5 days, observing colony morphology, measuring colony diameter and recording colony shape and color; the morphology was observed with a microscope. After 5 days of culture, trichoderma has a falling diameter of 8.5cm, hypha grows rapidly and is white villus, and the color of the front side and the back side is similar. The colony morphology is shown in FIG. 1. The hyphae were long and transverse and the microscopic morphology was seen in FIG. 2.

Example 5: influence of carbon source on the growth of trichoderma strains.

Starch, sucrose and carboxymethyl cellulose are respectively used as carbon sources, glucose in a PDA solid culture medium is replaced by equal amount, trichoderma strains are transferred to a flat plate and cultured for 5 days at 30 ℃, and trichoderma can grow on the culture medium and has similar morphology.

Example 6: molecular biological identification of Trichoderma strains.

1. Fungal DNA genome extraction

Taking the culture solution in the example 2, centrifuging, taking part of thalli, placing the thalli in a mortar, adding liquid nitrogen for full grinding, collecting the ground thalli in a centrifuge tube, and placing the centrifuge tube in a refrigerator at the temperature of minus 20 ℃ for standby.

200mg of liquid nitrogen ground thalli are taken, 3% CTAB is added, water bath is carried out at 65 ℃ for 45min, and centrifugation is carried out at 13000rpm for ten minutes. Taking supernatant and adding equal volume of phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1), and centrifuged at 13000rpm for ten minutes. The supernatant was taken and an equal volume of chloroform was added: isoamyl alcohol (24:1 by volume) was centrifuged at 13000rpm for ten minutes. Twice the volume of pre-chilled absolute ethanol was added to the supernatant, which was allowed to stand at-20℃for 20-30 minutes and centrifuged at 13000rpm for ten minutes. The supernatant was decanted, and the pellet was washed with 200. Mu.L of 70% ethanol and centrifuged at 13000rpm for ten minutes. Removing supernatant, and drying the precipitate at room temperature. Adding 30 μl of water to dissolve DNA completely, and storing in a refrigerator at-20deg.C.

PCR amplification

Taking the extracted strain DNA, and utilizing the sequence shown as SEQ ID NO:2, primer ITS1

(5'-TCCGTAGGTGAACCTGCGG-3') and the sequence of which is shown in SEQ ID NO:3 (5'-GCTGCGTTCTTCATCGATGC-3') and performing PCR amplification. The PCR reaction system is as follows: 25. Mu.L of 2 xTAQ enzyme, 2. Mu.L of ITS1 primer, 2. Mu.L of ITS2 primer, 5. Mu.L of template and 16. Mu.L of sterile water. The PCR reaction conditions were: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 60s, elongation at 72℃for 60s,30 cycles; extending at 72deg.C for 7min, and storing at 4deg.C.

rDNA-ITS sequencing and analysis

The target PCR product is recovered and purified by using a DNA gel rapid purification kit (Beijing full gold biotechnology Co., ltd.), and the purified PCR product is sent to Shanghai Biotechnology engineering service Co., ltd for sequencing, and the gene sequence is shown as SEQ ID NO: 1.

The sequences were aligned in NCBI by BLAST, analyzed using Mega 4.0 software and phylogenetic tree was constructed using the Neighbor-Joining method, as shown in FIG. 3.

The strain was found to have extremely high relatedness to Trichoderma strains by phylogenetic trees, and was identified as Trichoderma sp of the sarcodaceae family by binding morphological features.

The strain CY5104-1 is sent to China center for type culture collection for collection, and the collection number is CCTCC NO: m20221323, the preservation date is 2022, 8 and 24 days, and the preservation unit address is Wuhan university.

Example 7: influence of temperature on lactase enzyme Activity

1. Optimal reaction temperature of lactase

The crude enzyme solution was diluted appropriately, and enzymatic hydrolysis was performed at 20, 30, 37, 43, 50, 60, 70, and 80℃respectively, and lactase enzyme activity was measured as in example 2. And calculating the relative enzyme activities at different temperatures by taking the enzyme activity measured at the optimal reaction temperature as 100%. The relative enzyme activities are shown in the following table:

temperature/. Degree.C	20	30	37	43	50	60	70	80
									Relative enzyme activity/%	12.3	19.9	24.4	47.2	56.5	100	19.8	9.98

The relative enzyme activity change curve is shown in FIG. 4. The lactase expressed by Trichoderma has the highest activity at a reaction temperature of 60 ℃.

2. Thermostability of lactase

The crude enzyme solution is taken and properly diluted, and is respectively subjected to heat preservation treatment for 30 minutes at 20, 30, 37, 43, 50, 60, 70 and 80 ℃, and then is immediately placed in an ice bath for cooling, and the lactase enzyme activity is measured under the optimal reaction condition. The relative enzyme activities at different temperatures were calculated with the enzyme activities measured at different temperatures without incubation at 100%. The relative enzyme activities are shown in the following table:

temperature/. Degree.C	20	30	37	43	50	60	70	80
									Relative enzyme activity/%	85.6	90.4	100	96.6	97.6	69.9	20.4	16.4

The relative enzyme activity change curve is shown in FIG. 4. The lactase expressed by trichoderma has good heat stability in the range of 20-50 ℃ and the relative enzyme activity is higher than 85%.

Example 8: influence of pH on lactase enzyme Activity

1. Optimal reaction pH of lactase

The crude enzyme solution was diluted appropriately, and enzymatic hydrolysis was performed at the optimum reaction temperature under the conditions of pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively, and lactase enzyme activity was measured as in example 2. The relative enzyme activities at different pH values were calculated with the enzyme activities measured at the optimum reaction pH being 100%. The relative enzyme activities are shown in the following table:

pH	3.0	4.0	5.0	6.0	7.0	8.0	9.0	10.0
									relative enzyme activity/%	72.0	100	89.8	51.0	29.2	23.8	23.5	24.2

The relative enzyme activity change curve is shown in FIG. 5. The lactase expressed by trichoderma has the highest activity at the reaction pH of 4 and the activity at the pH of 3-5 is more than 70%.

Example 9: influence of temperature on protease activity

1. Optimal reaction temperature of protease

The crude enzyme solutions were diluted appropriately, and subjected to enzymatic hydrolysis at 20, 30, 37, 43, 50, 60, 70, and 80℃respectively, and protease activity was measured in the same manner as in example 3. And calculating the relative enzyme activities at different temperatures by taking the enzyme activity measured at the optimal reaction temperature as 100%. The relative enzyme activities are shown in the following table:

temperature/. Degree.C	20	30	37	43	50	60	70	80
									Relative enzyme activity/%	35.7	41.3	82.5	91.3	100	93.8	47.5	0

The relative enzyme activity change curve is shown in FIG. 6. The protease expressed by trichoderma has the highest activity at the reaction temperature of 50 ℃ and the activity of more than 80 percent in the range of 37-60 ℃.

2. Thermal stability of proteases

The crude enzyme solution is taken and properly diluted, and is respectively subjected to heat preservation treatment for 30 minutes at 20, 30, 37, 43, 50, 60, 70 and 80 ℃, and then is immediately placed in an ice bath for cooling, and the protease activity is measured under the optimal reaction condition. The relative enzyme activities at different temperatures were calculated with the enzyme activities measured at different temperatures without incubation at 100%. The relative enzyme activities are shown in the following table:

temperature/. Degree.C	20	30	37	43	50	60	70	80
									Relative enzyme activity/%	72.5	100	85.5	87.0	82.6	56.6	40.8	0

The relative enzyme activity change curve is shown in FIG. 6. The protease expressed by the trichoderma has good thermal stability in the range of 20-50 ℃ and the relative enzyme activity is higher than 70%.

Example 10: influence of pH on protease Activity

1. Optimal reaction pH of protease

The crude enzyme solution was diluted appropriately, and subjected to enzymatic hydrolysis at the optimum reaction temperature under the conditions of pH of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0, respectively, and the protease activity was measured in the same manner as in example 3. The relative enzyme activities at different pH values were calculated with the enzyme activities measured at the optimum reaction pH being 100%. The relative enzyme activities are shown in the following table:

pH	1.0.	2.0	3.0	4.0	5.0	6.0	7.0
								relative enzyme activity/%	0	100	87.0	85.8	41.7	45.6	31.3

The relative enzyme activity change curve is shown in FIG. 7. The protease expressed by Trichoderma has the highest activity at a reaction pH of 2 and has an activity of more than 85% at a pH of 2-4.

The above is merely exemplary embodiments of the present invention, and the scope of the present invention is not limited in any way. All technical schemes formed by adopting equivalent exchange or equivalent substitution fall within the protection scope of the invention.

Claims

1. A trichoderma strain capable of expressing lactase and protease, characterized in that: the strain is a Trichoderma (Trichoderma sp.) strain, the preservation date is 2022, 8 months and 24 days, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: m20221323.

2. The trichoderma strain capable of expressing lactase and protease according to claim 1, wherein the nucleotide sequence is shown in SEQ ID NO: 1.

3. Use of a strain of trichoderma capable of expressing lactase and protease as claimed in claim 1 or 2 for expressing lactase and/or protease.

4. The use of a trichoderma strain capable of expressing lactase and protease according to claim 3, wherein the optimal reaction temperature of lactase enzyme activity expressed by the trichoderma strain is 60 ℃.

5. The use of a trichoderma strain capable of expressing lactase and protease according to claim 4, wherein the lactase expressed by the trichoderma strain has good heat stability at 20-50 ℃.

6. The use of a trichoderma strain capable of expressing lactase and protease according to claim 5, wherein the lactase enzyme activity expressed by the trichoderma strain has an optimal reaction pH of 4.

7. The use of a trichoderma strain capable of expressing lactase and protease according to claim 3, wherein the protease activity expressed by the trichoderma strain has an optimal reaction temperature of 50 ℃.

8. The use of a trichoderma strain capable of expressing lactase and protease according to claim 7, wherein the protease expressed by the trichoderma strain has good thermal stability at 20-50 ℃.

9. The use of a trichoderma strain capable of expressing lactase and protease according to claim 8, wherein the protease activity expressed by the trichoderma strain has an optimal reaction pH of 2.