A tropical bacillus CSN-15 strain and its application

Abstract

The present invention belongs to the field of bioengineering technology, and provides a strain of microbial strains producing chitosanase, and particularly relates to a tropical bacillus, and a method for producing chitosanase using the strain and producing chitosan oligosaccharides using the enzyme. The present invention further studies the fermentation production of chitosanase and the method for preparing chitosan oligosaccharides by screening sea mud samples and obtaining chitosanase-producing strain CSN‑15. Compared with the currently commonly used chitosanase, the chitosanase produced by the CSN‑15 strain has a higher activity, an enzyme activity of 4.28U / ml, and can be applied to the degradation of chitosanase, has a shorter catalytic time, a wider range of applications, and is suitable for the industrial production of chitosan oligosaccharides.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and provides a microbial strain producing chitosanase, in particular to a tropical bacillus, and a method for producing chitosanase by using the strain and chitosan oligosaccharide by using the enzyme. Background Art

[0002] Chitosan is the only natural alkaline polysaccharide, but because of its large molecular weight and poor solubility in water, its development and utilization are severely restricted. With the research on chitosan, it is found that chitosan oligosaccharides prepared with chitosan as raw material have anti-inflammatory, anti-cancer, anti-tumor effects, and have good applications in many fields such as food, medicine, health products, and chemical industry. Chitosan oligosaccharides, also known as chitosan oligosaccharides, are oligosaccharide products with a polymerization degree between 2-20 obtained by degradation of chitosan. Compared with chitosan, chitosan oligosaccharides have better water solubility, higher biological activity, and are easier to be absorbed and utilized by organisms. Traditional preparation methods for chitosan oligosaccharides include physical methods and chemical methods. The physical method is relatively simple to operate and easy to control, and its main applications include ultrasonic degradation method, microwave degradation method, etc. However, although the physical method for preparing chitosan oligosaccharides has a mild reaction and simple operation, the production efficiency is relatively low, and chitosan oligosaccharides with the target polymerization degree cannot be obtained, which is also easy to cause waste of raw materials. The chemical method mainly uses various reagents to achieve the purpose through chemical reactions. It is the most commonly used and widely used method for the initial preparation of chitosan oligosaccharides. According to the different reagents selected, the chemical method can be divided into oxidative degradation method and acid degradation method. However, the oxidative degradation method not only affects the appearance of the product, but also brings a certain concentration to the purification and optimization of the subsequent product. The high temperature and high pressure reaction conditions in the acid degradation method are too harsh and difficult to control, which seriously affects the application of this method.

[0003] With the continuous development of glycobiology, chitosan oligosaccharides are further developed and utilized, and have research value in the fields of agriculture, food, medicine and medical materials, bringing great economic benefits. In recent years, research on chitosan oligosaccharides obtained by chitosan microbial fermentation and enzymatic hydrolysis has also been very active at home and abroad. At present, when preparing chitosan oligosaccharides, researchers mostly use enzymatic hydrolysis of chitosan because this reaction has the characteristics of high efficiency, uniform product and no pollution. However, due to the lack of development of high-activity, high-stability and low-cost chitosanase, the commercial demand for high-activity chitosanase cannot be met, and the development of this industry has been subject to certain restrictions. Therefore, it is particularly important to develop a method for producing chitosanase with high activity, high stability and low cost. Summary of the invention

[0004] In view of the shortcomings of the above-mentioned technology, the present invention obtains a chitosanase-producing strain CSN-15 by screening sea mud samples. The strain can produce chitosanase with higher activity and is applied to the degradation of chitosanase.

[0005] A tropical Bacillus (Bacillus tropicus) CSN-15 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC 24948.

[0006] Acquisition of Bacillus tropicus CSN-15:

[0007] The sea mud samples were collected in Yanwei Port, Haizhou Bay, Lianyungang (34°29′0″N, 119°47′0″E). 1 g of sea mud sample was weighed and added to 9 mL of sterile water. The sample was stirred with a glass rod for about 20 min to fully mix the sample with the sterile water. The sample was then diluted 10 times in a gradient. -4 , 10 -5 , 10 -6 dilution gradient;

[0008] Pour the dilution onto the screening medium and spread three plates for each concentration. Culture at 28°C for 7 days and observe whether there is a color change zone around the colonies on the screening medium and the growth of the colonies;

[0009] The strain with the best growth potential was selected as the object of subsequent research and named CSN-15. It was streaked and isolated and purified on LB medium plates for multiple times.

[0010] A single colony was inoculated on a slant medium and cultured in pure form. After culturing in a 28°C incubator for 2 days, it was moved to a -80°C refrigerator for storage.

[0011] The tropical bacillus CSN-15 obtained by the invention is used for fermentation production of chitosanase.

[0012] The present invention provides a method for using the obtained tropical Bacillus CSN-15 to ferment and produce chitosanase, and the specific steps are as follows:

[0013] (1) Activation of strains: The tropical Bacillus CSN-15 stored at -80°C was streaked onto LB solid medium for activation;

[0014] (2) Preparation of seed culture fluid: Under sterile conditions, the activated strain is selected and cultured on a seed culture medium to obtain a seed culture fluid;

[0015] (3) Fermentation to produce enzyme: The seed culture solution is inoculated into the fermentation medium for fermentation culture. The supernatant is collected by centrifugation, which is the crude enzyme solution of chitosanase.

[0016] The activation condition of the strain in step (1) is activation at 28° C. for 24 hours.

[0017] The seed culture conditions in step (2) are shaking flask culture at 28° C. and 180 r / min for 12 to 18 hours.

[0018] The inoculation ratio in step (3) is 5% (V / V), and the fermentation conditions for enzyme production are 28° C. and 180 rpm for 48 hours.

[0019] The method for producing chitosan oligosaccharide by hydrolyzing chitosan with the chitosanase obtained by the present invention is as follows: 1 ml of crude chitosanase solution is mixed with 50 ml of 12% colloidal chitosan solution, and reacted in a 35°C water bath for 60 minutes to completely degrade chitosan to obtain chitosan oligosaccharide.

[0020] Preferably, the preparation method of the 12% colloidal chitosan solution is: dissolve 12g of chitosan powder in 90ml of 2% acetic acid solution, add an appropriate amount of alkaline solution (1% sodium acetate solution), correct the pH to 5.0, and add water to a volumetric flask to 100ml, which is 12% colloidal chitosan.

[0021] The invention has the following beneficial effects: compared with the currently commonly used chitosanase, the chitosanase produced by the CSN-15 strain has a higher activity, with an enzyme activity of 4.28U / ml. The chitosanase has an optimum pH of 5.0 and an optimum temperature of 35°C, and the amount of chitosan added has no effect on the activity of the enzyme. In addition, the enzyme has a short catalytic time, and the chitosan oligosaccharide yield obtained in 1 hour is high, accounting for 83% of the total product. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :Screening culture medium transparent zone

[0023] Figure 2 :Glucose standard curve

[0024] Figure 3 :SDS gel electrophoresis and activity staining, 1: Marker; 2: blank group; 3: experimental group (without chitosan); 4: experimental group (with chitosan); 5: activity staining

[0025] Figure 4 :Effect of pH on the activity of CSN-15 chitosanase

[0026] Figure 5 :Effect of temperature on the activity of CSN-15 chitosanase

[0027] Figure 6 : Results of chitosan hydrolysis by CSN-15 chitosanase, A is the control group (without crude enzyme solution), B is the experimental group (with crude enzyme solution)

[0028] Figure 7 :Thin layer chromatography results of chitosan enzymatic hydrolysis products DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] Example 1 Strain Screening

[0031] The sea mud samples were collected in Yanwei Port, Haizhou Bay, Lianyungang (34°29′0″N, 119°47′0″E). 1 g of sea mud sample was weighed and added to 9 mL of sterile water. The sample was stirred with a glass rod for about 20 min to fully mix the sample with the sterile water. The sample was then diluted 10 times in a gradient. -4 , 10 -5 , 10 -6 After different dilutions, pour the dilution onto the screening medium, and apply three plates for each concentration. Culture at 28°C for 7 days, and observe whether there is a color change circle around the colony on the screening medium and the growth of the colony. First, pick the strains that grow normally on the culture medium and produce a color change circle, and perform multiple streaking isolation and purification culture on the LB culture medium plate. Inoculate a single colony on the LB culture medium slope and perform pure culture. After culturing in a 28°C incubator for 2 days, move it to a -80°C refrigerator for storage. Among them, select the color change circle ( Figure 1 The strain with the largest size and the best growth potential (as shown) was selected as the object of subsequent research and named CSN-15.

[0032] Screening culture medium: solution A (1 g chitosan), solution B (0.2 g KH2PO4, 0.4 g K2HPO4, 0.14 g MgSO4, 0.2 g NaCl, 1 g (NH4)2SO4, 4 g agar), sterilized at 120°C for 20 min, and then solution A and solution B were mixed under sterile conditions.

[0033] LB medium: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, 2% agar powder, sterilized at 120°C for 20 min, and then cooled to room temperature.

[0034] Example 2 Bacterial species identification

[0035] 16S rDNA identification of CSN-15 strain: Pick the target colony and inoculate it into LB liquid culture medium, culture at 37℃, 180rpm, and culture for 6-8h. Collect 0.5ml of bacterial culture solution in a 1.5ml centrifuge tube, centrifuge at 12000r / min for 2min, and discard the supernatant. Use a rapid extraction kit to extract the DNA of the strain CSN-15 genome, and use it as a template, using universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and

[0036] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3', for amplification of 16S rDNA gene.

[0037] LB liquid culture medium: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, sterilized at 120°C for 20 min, and then cooled to room temperature.

[0038] PCR amplification conditions: pre-denaturation at 94°C for 8 min; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, for a total of 28 cycles; extension at 72°C for 5 min.

[0039] PCR results: After obtaining the corresponding PCR product, the product was sent to the biological company for sequencing to obtain the 16S rDNA sequence. The obtained sequence was uploaded to the database GenBank (GenBank NO.: ON422091.1), the sequencing results were analyzed by BLAST, and the strain sequence with higher homology was selected to compare with the strain CSN-15 to determine the type of the strain. The strain was determined to be Bacillus tropicus through the 16S rDNA full sequence, and has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit date of and a deposit number of CGMCC 24948.

[0040] Example 3 Fermentation enzyme production

[0041] The tropical Bacillus CSN-15 preserved at -80°C was streaked onto LB solid culture medium with three zones, and cultured in a constant temperature box at 28°C for 24 hours. Then, a single colony was picked and inoculated into LB liquid culture medium, and cultured for 24 hours to obtain a seed culture solution. The seed culture solution was inoculated into LB liquid culture medium at a ratio of 5% (V / V), and fermented at 28°C, 180rpm, for 48 hours to obtain a fermentation solution rich in chitosanase.

[0042] LB liquid culture medium: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, sterilized at 120°C for 20 min, and then cooled to room temperature.

[0043] LB solid medium: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, 2% agar. Sterilize at 120℃ for 20min and then cool to room temperature.

[0044] Example 4 Enzyme activity determination

[0045] Chitosanase activity detection reference "3,5 dinitrosalicylic acid method (DNS) to measure chitosanase activity" published in the method, according to the DNS method to obtain glucose standard curve as follows Figure 2As shown, the linear equation is: y = 1.2681x-0.0235, R 2 The standard curve was used to measure the chitosanase activity in the fermentation broth, and the strain with the highest chitosanase activity was 4.28 U / ml.

[0046] Example 5 SDS-PAGE gel electrophoresis and activity staining

[0047] Prepare samples: 20 μL of chitosanase-rich fermentation broth sample was thoroughly mixed with 5 μL of 5× protein loading buffer (purchased from Beijing Solebao Technology Co., Ltd.), centrifuged, and treated in a boiling water bath for 10 min. Spotting: 5 μL of Rainbow Protein Marker (10-180 kDa, purchased from Beijing Solebao Technology Co., Ltd.) and each protein sample were spotted in sequence. Gel running: The protein electrophoresis voltage started from 80 V, and the constant voltage was 140 V when the band entered the separation gel. When the band ran to the bottom, the electrophoresis ended and the gel was taken. Color development: After the electrophoresis, it was stained with Coomassie Brilliant Blue solution for 30 minutes, and the staining solution was discarded and the residual staining solution on the surface of the gel was rinsed with distilled water. Decolorization: Add decolorizing solution, boil in a microwave oven, and then change the decolorizing solution. Repeat 3-4 times, add decolorizing solution, and decolorize overnight at 60 rpm until the band is clear.

[0048] Replace the double distilled water component in the SDS-PAGE separation gel with 0.1g / 100mL chitosan. After the electrophoresis, wash the separation gel in a refolding buffer (50mmol / L KH2PO4-NaOH (pH6.5), 1g / 100mL TritonX-100) overnight (at least 10-12h); then place the separation gel in a 10mmol / L acetic acid-sodium acetate solution (pH5.5) and keep it at 45℃ for 6-8h. Finally, stain with 0.1g / 100mL Congo red for 15-25min. After staining, wash off the residual dye with distilled water, and then decolorize in 1mol / L NaCl solution, and change the decolorizing solution every 2h, and decolorize overnight at 60rpm until the bands are clear.

[0049] SDS-PAGE gel electrophoresis was used to detect the purity and molecular weight of chitosanase obtained by fermentation of strain CNS-15. The presence of chitosanase was confirmed by zymogram analysis. The zymogram showed that it had chitosanase activity. The results are as follows: Figure 3 It was shown that the purified protein peak was the chitosanase activity peak, and the molecular weight of the purified pure enzyme was about 37 kDa.

[0050] Example 6 Enzymatic Properties

[0051] Optimum pH

[0052] The crude enzyme solution of strain CSN-15 was tested for enzyme activity at different pH values. A 1% colloidal chitosan solution was prepared with buffer, and its pH was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and mixed with the crude enzyme solution at a volume ratio of 1:1 to test the enzyme activity. 1% colloidal chitosan: Add 1g chitosan to about 10-20mL acetic acid solution, stir until dissolved, add 60-70mL distilled water, and adjust the pH to 5.0 with 2mol / L NaOH.

[0053] Depend on Figure 4 It can be seen that the optimum pH is 5, and about 80% of the enzyme activity is still retained between pH 3-6, indicating that the enzyme is acidic chitosanase and meets the requirements of industrial applications.

[0054] Optimum temperature

[0055] The crude enzyme solution of strain CSN-15 was tested for enzyme activity at different temperatures. 20, 25, 30, 35, 40, 45, 50, 55, and 60°C were selected as reaction temperatures, and the crude enzyme solution and 1% colloidal chitosan were reacted at a volume ratio of 1:1:1 for 15 minutes to test the enzyme activity.

[0056] Depend on Figure 5 It can be seen that the optimum temperature is 35°C, and the enzyme activity still maintains 90% at a temperature between 30-45°C, indicating that the enzyme has a good temperature range.

[0057] Example 7 Enzyme hydrolysis experiment

[0058] 1 ml of the enzyme solution and 50 ml of 12% colloidal chitosan were placed in a shaker at 35°C and 160 r / min for 1 hour to react, and a control group and an experimental group were set up.

[0059] The colloidal chitosan solution was prepared by dissolving 12 g of chitosan powder in 90 ml of 2% acetic acid solution, adding an appropriate amount of alkaline solution (1% sodium acetate solution), adjusting the pH to 5.0, and adding water to a volumetric flask to 100 ml, which is 12% colloidal chitosan.

[0060] according to Figure 6 Observing the degree of hydrolysis, the method of obtaining chitosan in industry is acid hydrolysis, which makes the enzyme capable of hydrolyzing chitosan have a certain acid resistance, but the short half-life under this condition is the bottleneck restricting the enzymatic preparation of chitosan oligosaccharides. The enzyme has strong hydrolysis activity under acidic conditions (PH≤5) and hydrolyzes colloidal chitosan within 1 hour, and has a certain acid resistance.

[0061] Analysis of components of chitosanase hydrolysis products

[0062] The supernatant of the enzymatic hydrolysis product was removed of denatured precipitated proteins, then washed with anhydrous ethanol, centrifuged after standing for 12 hours, and washed three times. The obtained precipitate was freeze-dried to obtain powdered chitosan oligosaccharide. TM The system (Dalian Institute of Chemical Physics, Chinese Academy of Sciences) was used to qualitatively analyze the components of the enzymatic hydrolysis products. The Data Explorer software processed the raw data and drew mass spectra for analysis. The abscissa of the mass spectrum represents the mass-to-charge ratio (m / z) of the ion, and the value represented by the abscissa is the mass of the ion. The ordinate represents the intensity of the ion flow (test conditions: Laser Intensity: 5000, Min S / N: 100, Mass Tolerance: + / 0.5m / z, Min Peaks to March: 5, MALDI Matrix: DHB, Total Shots / Spectrum: 1000).

[0063] like Figure 7 As shown in the figure, when the CSN-15 chitosanase hydrolysis product was determined, the product contained glucosamine, indicating that the enzyme was an exo-chitosanase; the high-resolution mass spectrometry analysis of the enzymatic hydrolysis product showed that the relative molecular mass of chitosan oligosaccharides was between 399 and 3009, and the distribution of chitosan oligosaccharides was relatively wide. According to the molecular weight, chitosan oligosaccharides with a degree of polymerization of 2 to 10 were distributed in the enzymatic hydrolysis product, accounting for 83% of the total product. The recombinant chitosanase obtained in this study is a conventional chitosanase, which is specific to the degradation of chitosan.

Claims

1. A tropical bacillus ( Bacillus tropicus )CSN-15 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC 24948.

2. The tropical Bacillus CSN-15 as claimed in claim 1 is used for fermentation production of chitosanase.

3. The method for fermenting and producing chitosanase using tropical Bacillus CSN-15 as claimed in claim 1, comprising the following steps: (1) Activation of strains: Striking three zones of tropical Bacillus subtilis CSN-15 stored at -80°C onto LB solid medium for activation; (2) Preparation of seed culture medium: Under sterile conditions, the activated strain is selected and cultured on a seed culture medium to obtain a seed culture medium; (3) Fermentation to produce enzymes: The seed culture solution is inoculated into the fermentation medium for fermentation culture. The supernatant is collected by centrifugation, which is the crude enzyme solution of chitosanase.

4. The method according to claim 3, characterized in that The activation condition of the strain in step (1) is activation at 28° C. for 24 hours.

5. The method according to claim 3, characterized in that The seed culture conditions in step (2) are shaking flask culture at 28°C and 180 r / min for 12 to 18 hours.

6. The method according to claim 3, characterized in that The inoculation ratio in step (3) is 5%, and the fermentation conditions for enzyme production are 28° C. and 180 rpm for 48 hours.

7. The chitosanase obtained by fermentation production according to the method of claim 3 is used for hydrolyzing chitosan to produce chitooligosaccharides.

8. A method for hydrolyzing chitosan by chitosanase obtained by fermentation production according to the method of claim 3, comprising the following steps: 1 ml of crude chitosanase solution was mixed with 50 ml of 12% colloidal chitosan solution and reacted in a 35°C water bath for 60 min to completely degrade chitosan and obtain chitooligosaccharides.

9. The method according to claim 8, characterized in that The preparation method of the 12% colloidal chitosan solution is as follows: dissolve 12g of chitosan powder in 90ml of 2% acetic acid solution, add an appropriate amount of 1% sodium acetate solution, correct the pH to 5.0, and add water to a volumetric flask to 100ml to obtain 12% colloidal chitosan.

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