CN112159782A

CN112159782A - Bacillus subtilis strain SH21 for producing chitinase, method for producing chitinase, microbial preparation and application

Info

Publication number: CN112159782A
Application number: CN202011172383.2A
Authority: CN
Inventors: 谢远红; 张红星; 庞远祥; 金君华; 刘慧�
Original assignee: Beijing University of Agriculture
Current assignee: Beijing University of Agriculture
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-01-01
Anticipated expiration: 2040-10-28
Also published as: CN112159782B

Abstract

The invention provides a bacillus subtilis strain SH21 for producing chitinase, a method for producing the chitinase, a microbial preparation and application. The Bacillus subtilis strain SH21 has chitinase-producing activity. The chitinase activity of the bacillus subtilis strain can reach 4.02U/mL after fermentation, and the chitinase purification times are improved by 7.23 times and the specific activity is 154.5U/mg after a fermentation product is purified by the combination of salting out, gel filtration chromatography and ion exchange chromatography. The invention also provides application of the bacillus subtilis strain or the microbial preparation in inhibiting growth of fusarium solani.

Description

Bacillus subtilis strain SH21 for producing chitinase, method for producing chitinase, microbial preparation and application

Technical Field

The invention relates to the technical field of microorganisms, in particular to a Bacillus subtilis SH21 strain, a method for producing chitinase, a microbial preparation and application.

Background

Plant diseases are one of the natural disasters that seriously jeopardize agricultural production. According to reports from the food and agriculture organization (grain agriculture organization) in the united nations, there is a worldwide loss of more than 10% of grain and cotton production annually due to disease. Plant diseases not only cause the yield reduction of crops, but also seriously threaten the quality and safety of agricultural products to a certain extent. Currently, control of these diseases relies heavily on the use of synthetic chemical fungicides. However, the use of chemical fungicides has adverse effects on the environment and human health. Worse still, long-term use of chemical fungicides may lead to drug resistance. Therefore, there is an urgent need for safer, more ecologically friendly alternative fungicides to control plant diseases.

Chitinase has biological activities of bacteriostasis, cancer resistance, cholesterol reduction, blood pressure reduction and the like, is called as the sixth life element following five life elements of sugar, protein, fat, vitamins, mineral substances and the like by modern science, is widely applied to industries such as medicine, food, chemical industry, cosmetics and the like, and has very high application value and development prospect. Thus, the preparation and development of chitinase have received increasing attention and interest. The production of chitinase by microbial techniques is one of the important routes for producing chitinase. There are various microorganisms such as Enterobacter bacteria, Streptomyces actinomycetes, etc. which can secrete chitinase, however, fermentation conditions for preparing chitinase are different among different strains, and the chitinase of the strains has greatly different enzyme yield and enzyme activity.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a Bacillus subtilis SH21 for producing chitinase, a method for producing the chitinase by using the Bacillus subtilis, a microbial preparation and application. The bacillus subtilis strain has high chitinase yield and activity, and the chitinase activity can reach 4.02U/mL after fermentation.

The technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a chitinase-producing Bacillus subtilis strain SH21, which is preserved in 2019 at 23.9.9 with the preservation units as follows: china general microbiological culture Collection center, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation number is CGMCC NO. 18612.

In a second aspect, the present invention provides a method of producing chitinase, the method comprising: carrying out fermentation culture by using the strain SH 21; the fermentation medium comprises the following components: maltose 2g, casein peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g and 1000mL of distilled water.

In a particular embodiment, the temperature of the fermentation culture is 20 ℃ to 40 ℃, preferably 29 ℃ to 30 ℃; the fermentation time is 24-60h, preferably 48 h;

preferably, the initial pH of the fermentation culture is 5-8, more preferably pH 7.

In one embodiment, the method further comprises inoculating the strain prior to fermentation into a seed medium having a composition of: 10g of peptone, 5g of yeast extract powder, 10 mL of NaCl10g and 1000mL of distilled water;

the inoculum size of the inoculation is 1% -3% v/v, preferably 1.5% -2.5% v/v, more preferably 2% v/v.

In another embodiment, the method further comprises the step of performing chitinase purification after the fermentation culture:

the purification is a combined purification by salting out, gel filtration chromatography and ion exchange chromatography.

In another embodiment, the salting out is performed with ammonium sulfate, preferably 70% to 80% ammonium sulfate, more preferably 75% ammonium sulfate;

the chromatographic column of the gel filtration chromatography is a Superdex 75PG chromatographic column;

the chromatographic column of the ion exchange chromatography is SPnocurose HPR.

In another embodiment, the method further comprises the identification of the fermentation product chitinase.

Preferably, the identification includes identification of molecular weight, purity and amino acid of the chitinase.

In some specific embodiments, the purity is identified using high performance liquid chromatography.

In some specific embodiments, the amino acid is identified using mass spectrometry.

In a third aspect, the present invention also provides a fermentation product obtained by the above process.

In a fourth aspect, the present invention provides a microbial preparation comprising the aforementioned chitinase-producing Bacillus subtilis strain SH21 or a fermentation product comprising the aforementioned.

In a fifth aspect, the present invention also provides the use of the bacillus subtilis strain or the microbial preparation for inhibiting the growth of fusarium solani.

In some embodiments, the inhibition of fusarium solani growth inhibition assay is performed using the tube-plate method.

The invention provides a Bacillus subtilis SH21 for producing chitinase, which is preserved in 2019 at 23.9.18612 with the preservation number of CGMCC NO.18612 and the preservation unit of: china general microbiological culture Collection center, address: the Xilu No.1 Hospital No. 3, Beijing, Chaoyang, was tested by the Collection as a viable strain and deposited at 18 days 11 and 2019.

According to the invention, a strain of bacillus subtilis producing chitinase is screened, and the chitinase activity in the supernatant obtained by fermenting the bacillus subtilis SH21 is 1.12U/mL.

The invention further provides a method for producing chitinase, which comprises optimizing a fermentation culture medium and fermentation conditions, wherein the activity of the chitinase after fermentation can reach 4.02U/mL; after the fermentation product is further purified by the combination of salting out, gel filtration chromatography and ion exchange chromatography, the purification multiple of the chitinase is improved by 7.23 times, and the specific activity is 154.5U/mg. The purified chitinase was homogeneous as confirmed by SDS-PAGE, and the molecular weight was about 30kDa as identified by SDS-PAGE and mass spectrometry. The purity of the purified chitinase reaches 91.33%.

The bacillus subtilis strain SH21 and the microbial preparation containing the bacillus subtilis strain or the fermentation product thereof can inhibit the growth of fusarium solani, thereby reducing plant diseases and insect pests.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the effect of inoculum size on the production of enzyme by Bacillus subtilis SH 21;

FIG. 2 is a graph showing the effect of fermentation temperature on the production of enzyme by Bacillus subtilis SH 21;

FIG. 3 is a graph showing the effect of fermentation time on the production of enzyme by Bacillus subtilis SH 21;

FIG. 4 is a graph showing the effect of initial pH on the production of enzyme by Bacillus subtilis SH 21;

FIG. 5 is a diagram of a protein purification process;

FIG. 6 is an SDS-PAGE pattern of purified proteins;

FIG. 7 is a diagram showing purity identification;

FIG. 8 is a protein mass spectrum;

FIG. 9 shows different saturation levels (NH)₄)₂SO₄A precipitated protein bacteriostatic map;

FIG. 10 shows the effect of carbon source on the production of enzyme by Bacillus subtilis SH 21;

FIG. 11 shows the effect of nitrogen source on the production of enzyme by Bacillus subtilis SH 21.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the formulations of chitinase-producing screening medium, LB medium, described in the following examples are as follows:

chitinase production screening culture medium: chitin powder 0.2%, K₂HPO₄ 0.07％，KH₂PO₄0.03％，MgSO₄0.05%, agar 2.0%, pH 7.0.

The LB medium formula: peptone 10g, yeast extract g, NaCl10g, (solid medium plus 15g agar) distilled water 1000mL, pH 7.0. Seed liquid culture medium: the same as LB culture medium. Example 1 selection and isolation of Bacillus subtilis SH21 and detection of chitinase Activity

The purpose of this example is to isolate and purify Bacillus subtilis from fermented soybean paste for screening strains with high chitinase activity.

The specific experimental method is as follows:

(1) grinding soybean paste obtained from natural fermentation of Sichuan Daliangshan farmers, dipping a small amount of sauce powder in an inoculating loop, coating the sauce powder on a chitinase-producing screening culture medium plate, culturing at 30 ℃ for 1-2 days, observing colony growth, and selecting a transparent colony with good growth and large decomposition loop.

Inoculating the strain to LB solid culture medium, purifying until single strain is left, and preserving on inclined plane.

Inoculating the strain to a seed culture medium for culturing for 12h, then inoculating the strain to a fermentation culture medium in an inoculation amount of 2% (V/V), and culturing for 48h at 30 ℃ and 180 r/min. The enzyme activity of chitinase is determined after the fermentation liquor is centrifuged at 10000 Xg for 10min at 4 ℃.

The fermentation medium formula comprises: wheat (Haima Hayata, HaShoot sugar 2g, casein peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄0.7g，K₂HPO₄0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 7.0.

(2) Detection of chitinase activity in fermentation broth

Making a standard curve: accurately weighing 0.01g of paranitroaniline, adding water to dissolve the paranitroaniline, fixing the volume in a 100mL volumetric flask, uniformly mixing the paranitroaniline and the paranitroaniline, performing gradient dilution, and measuring the light absorption value (A400) of each dilution at 400nm by taking distilled water as a reference. And (3) drawing a standard curve by taking the concentration of the paranitroaniline as an abscissa and taking A400 as an ordinate.

And (3) determination of a sample to be tested: 1mL of the crude enzyme solution (supernatant from the fermentation in step (1)) was added with 1mL of 200mg/L p-nitroacetanilide and 3mL of a phosphate buffer, incubated at 30 ℃ for 15min, quenched in a boiling water bath, centrifuged, and then the absorbance of each tube was measured in a spectrophotometer (A400).

The experimental results are as follows:

1. in the step (1) of this example 1, 3 strains of Bacillus subtilis having a decomposition cycle were obtained by separation and purification, and the strain having the largest decomposition cycle was SH 21.

2. Standard curve: the result shows that the linear relation between y and absorbance in the concentration range of 0-10 mg/L of the paranitroaniline is 0.036x +0.008, and R2 is 0.9993, which indicates that the curve fitting degree is good.

3. The chitinase activity in the fermentation supernatant of the 3 strains is shown in the table 1, and the highest chitinase activity in the fermentation supernatant of the bacillus subtilis SH21 is 1.12U/mL. Table 1 chitinase activity in fermentation broths of three strains was compared.

TABLE 1

EXAMPLE 2 production of chitinase by Bacillus subtilis Strain SH 21- -fermentation conditions were optimized

The liquid fermentation conditions of the bacillus subtilis SH21 are optimized in the embodiment, and the liquid fermentation conditions comprise a single-factor optimization test and a response surface test. The specific test method is as follows:

inoculating bacillus subtilis SH21 into a seed culture medium, carrying out shaking culture at 30 ℃ and 180r/min for 12h, then transferring to a fermentation culture medium, and setting initial culture conditions as follows: the fermentation temperature was 37 ℃, the initial pH 7, the inoculum size was 2%, and the fermentation time was 24 h.

The main influencing factors (inoculation amount, fermentation temperature, fermentation time and initial pH value) of the fermentation culture are subjected to single-factor optimization test, and the enzyme activity of each fermentation liquid under different conditions is measured (the measuring method is shown in example 1). In 20mL of fermentation medium, the inoculum size was set to 1%, 2%, 3%, 4%, 5% (V/V), respectively; the fermentation temperature is set to 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃; the fermentation time is respectively set to 24h, 36h, 48h, 60h and 72 h; the initial pH values were set at 5.0, 6.0, 7.0, 8.0, 9.0. On the basis, the optimal range of each level is determined, and response surface test optimization is carried out.

The results of the one-factor optimization experiment are shown below:

(1) the effect of the inoculation amount on the strain SH21 is shown in figure 1, along with the increase of the inoculation amount, the relative enzyme activity firstly rises and then falls, the inoculation amount is high, the strain grows quickly, the nutrient consumption is accelerated, and when the inoculation amount is 2%, the relative enzyme activity is highest, so that the inoculation amount is determined to be 2%.

(2) The results of the effect of the fermentation temperature on the strain SH21 are shown in FIG. 2, the relative enzyme activity is increased and then decreased along with the increase of the fermentation temperature, the enzyme protein is denatured and inactivated when the temperature is high, and the maximum value is reached at 30 ℃, so the fermentation temperature is determined to be 30 ℃.

(3) The effect of fermentation time on the strain SH21 is shown in figure 3, and as the fermentation time is prolonged, nutrient substances in the culture medium are gradually consumed, relative enzyme activity firstly rises and then falls, and relative enzyme activity is highest in 48 hours.

(4) The effect of the initial pH value on the strain SH21 is shown in FIG. 4, as the initial pH value is increased, the relative enzyme activity is increased and then decreased, when the pH value is higher, the enzyme protein is denatured and inactivated, and the pH value of 7.0 is the highest relative enzyme activity, so the initial pH value of the culture medium is determined to be 7.0.

The results of the response surface test are shown below: after single factor screening, PB test and response surface optimization test, the influence of various factors on the enzyme activity is analyzed. The optimal optimization conditions according to comparative analysis of the regression model are shown in table 2. In actual experiments, the optimum conditions were maintained with slight modifications under appropriately set conditions. The optimal conditions are that the fermentation temperature is 29 ℃, and the fermentation time and the pH are respectively maintained at 48h and 7.0. Under these conditions, the chitinase activity was 4.02U/ml.

TABLE 2

Example 3: purification and characterization of chitinase, a fermentation product of Bacillus subtilis

This example is a purification and characterization of the chitinase enzyme, a fermentation product of Bacillus subtilis, as described above. The specific method comprises the following steps:

the enzyme was precipitated with 75% ammonium sulfate, dialyzed against 0.02M Tris-HCl buffer, and the dialysate was applied to a Superdex 75PG column (1.6X 64.6cm) pre-equilibrated with the same buffer, followed by elution at a flow rate of 1 mL/min. The elution peaks were collected, the enzyme activity and the protein concentration in each fraction were measured separately, and the fraction containing the highest active enzyme was collected, concentrated by ultrafiltration, and then loaded on a SP HPR column equilibrated in advance with PBS (pH 6.5), and then eluted with the same buffer containing 1mol of NaCl at a flow rate of 2.5 mL/min. Fractions with a single peak of fibrinolytic activity were pooled, concentrated and separated by SDS-PAGE. The protein purification process is shown in FIG. 5.

The protease yields and purification fold for each purification procedure are shown in Table 3. The purification multiple of the enzyme is 7.23 times, the protein recovery rate is 1.5 percent, and the specific activity is 154.5U/mg. The molecular weight of the enzyme was determined to be about 30kDa by mass spectrometry. The SDS-PAGE pattern is shown in FIG. 6, and a distinct protein band is obtained at about 30 kDa.

TABLE 3 enzyme yields and fold purification after each purification procedure

(2) Purity of the purified enzyme by HPLC

An UltimateXB-C4 chromatography column (250X 4.6mm, 5 μm) was used. The mobile phase was (a) 0.1% trifluoroacetic acid and (B) acetonitrile. The elution conditions were: linear gradient 0-5% B for 0-1 min; 1-25 min, linear gradient 5-95% B; 25-28 min, linear gradient 95-5% B. The flow rate was 1ml/min and the amount of sample was 10. mu.L. The column temperature was 25 ℃ and the wavelength was 214 nm. The results of the detection are shown in FIG. 7. Three absorption peaks occur at 13.230, 20.798 and 23.799min, respectively. The target protein has an absorption peak at 13.230min, has purity of 91.33% and can be used for further amino acid sequencing analysis.

(3) Amino acid identification

Identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fragment spectra were searched using the NCBI non-redundant protein database. A Maxquant search engine is used. The results of the identification are shown in FIG. 8, and the purified enzyme was used for analyzing the amino acid sequence. The resulting protein sequences were searched in the Maxquant database using BLAST and finally identified as chitinases.

Example 4: analysis of bacteriostatic properties of fermentation product chitinase

This example is an analysis of the bacteriostatic properties of the aforementioned chitinase, a fermentation product of Bacillus subtilis. The specific method comprises the following steps:

taking Fusarium solani as an indicator, vertically and lightly placing Oxford cups at different saturation (NH)₄)₂SO₄And (3) adding 200 mu L of bacterial liquid to be detected into the Oxford cup on the precipitated protein plate, diffusing at the low temperature of 4 ℃ for 2h, culturing at the temperature of 28 ℃ for 2-3 days, and observing the diffusion condition of hyphae. The results are shown in FIG. 9. When (NH)₄)₂SO₄The antibacterial effect is best when the saturation is 80%.

Comparative example 1:

the same as in example 1, except that the fermentation medium was:

glucose 2g, peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O 0.05g，1000mL of distilled water, pH 6, and culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 2:

the same as in example 1, except that the fermentation medium was:

lactose 2g, peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 3:

the same as in example 1, except that the fermentation medium was:

sucrose 2g, peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 4:

the same as in example 1, except that the fermentation medium was:

maltose 2g, peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 5

The same as in example 1, except that the fermentation medium was:

2g of soluble starch, 2g of peptone and MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

The relative enzyme activities of comparative examples 1-5 are shown in FIG. 10.

Comparative example 6

The same as in example 1, except that the fermentation medium was:

maltose 2g, caseinPeptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 7

The same as in example 1, except that the fermentation medium was:

maltose 2g, tryptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

Comparative example 8

The same as in example 1, except that the fermentation medium was:

maltose 2g, soybean peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g, distilled water 1000mL, pH 6, culture conditions of 37 ℃, 180rpm, 24 h.

The relative enzyme activities of comparative examples 6-8 are shown in FIG. 11.

From the above examples and comparative examples, it can be seen that the relative enzyme activities of the fermentation broths were optimized under the carbon source (maltose) and nitrogen source (casein peptone) of the present invention and the optimized culture conditions (inoculum size, fermentation temperature, fermentation time, pH) of the present invention.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A chitinase-producing Bacillus subtilis strain SH21, characterized in that the preservation unit of the strain is as follows: china general microbiological culture Collection center; the preservation number is CGMCC NO. 18612; the preservation time is 2019, 9 and 23 days.

2. A method for producing chitinase, which comprises carrying out fermentation culture using the strain SH21 according to claim 1; the fermentation medium comprises the following components: maltose 2g, casein peptone 2g, MgSO₄·7H₂O 0.5g，KH₂PO₄ 0.7g，K₂HPO₄ 0.3g，FeSO₄·7H₂O0.05g and 1000mL of distilled water.

3. The method according to claim 2, wherein the temperature of the fermentation culture is 20 ℃ to 40 ℃, preferably 29 ℃ to 30 ℃.

4. The method according to claim 2, wherein the fermentation time of the fermentation culture is 24-60h, preferably 48 h;

5. The method of claim 2, further comprising inoculating the strain to a seed medium prior to fermentation, the seed medium having a composition of: 10g of peptone, 5g of yeast extract powder, 10g of NaCl and 1000mL of distilled water;

6. The method of claim 2, further comprising the step of chitinase purification after the fermentation culture:

7. The method according to claim 6, wherein the salting out is performed with ammonium sulfate, preferably 70-80% ammonium sulfate;

8. A fermentation product obtained by the process of any one of claims 2 to 5.

9. A microbial preparation comprising the strain of claim 1 or comprising the fermentation product of claim 8.

10. Use of a bacillus subtilis strain according to claim 1 or a microbial preparation according to claim 9 for inhibiting the growth of fusarium solani.