CN113913450B - Method for expressing chitosanase by rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, fermentation bacteria and application - Google Patents

Abstract

The invention discloses a method for expressing chitosanase by using Rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, fermentation agent and application. The chitosanase gene sequence encoding Bacillus subtilis is The recombinant plasmid is constructed, and the recombinant plasmid is transformed into the body of Rhodopseudomonas palustris through Escherichia coli to obtain a recombinant strain of Rhodopseudomonas palustris. The recombinant strain of Rhodopseudomonas palustriae is fermented and cultured to obtain chitosanase. The method of using Rhodopseudomonas palustris to express chitosanase by constructing a recombinant plasmid containing a chitosanase gene and expressing it naturally in Rhodopseudomonas palustris does not require chitosan induction and does not require chitosan induction. Affecting the growth of recombinant bacteria, the expressed cellular chitosanase can continuously hydrolyze chitosan to form chitosan oligosaccharide, improve the solubility of chitosan, and fermentation bacteria containing chitosan oligosaccharide can be used directly.

Description

Methods for expressing chitosanase using Rhodopseudomonas palustris, chitosanase, recombinant plasmid Granules, recombinant bacteria, fermentation bacteria and their applications

Technical field

The present invention relates to the field of bioengineering technology, and in particular, to a method for expressing chitosanase using Rhodopseudomonas palustris. In addition, the invention also relates to a chitosanase, recombinant plasmid, recombinant bacteria, fermentation bacteria and applications.

Background technique

Chitosan is a product obtained by removing part of the N-deacetyl group from chitin. It has many characteristics and is widely used in the agricultural field. However, chitosan with a high degree of polymerization has a large molecular weight, poor water solubility, is not easily absorbed by plants, and has a strong flocculation effect, which seriously limits the direct use of chitosan and its compound use with other biological and microbial agents. Chitosanase (EC 3.2.1.132) is used to hydrolyze chitosan, which can reduce the degree of polymerization of chitosan and produce chitosan oligosaccharide, which greatly improves the many disadvantages of chitosan in use.

At present, chitosanase genes mainly come from bacterial and fungal microorganisms. However, chitosanase genes in microorganisms cannot be naturally expressed. Chitosan needs to be added to the culture medium to induce gene expression. However, chitosan is insoluble in water and needs to be dissolved by acetic acid or even hydrochloric acid. When chitosan colloidal solution is added to the culture medium, it seriously affects the pH of the culture medium and thus affects the growth of microorganisms. Secondly, the stability of chitosan colloidal solution is very poor, and it is easy to precipitate after being added to the culture medium. It also has a strong flocculating effect on microorganisms, further restricting the growth of microorganisms, resulting in no expression of chitosanase or only a small amount of expression.

Contents of the invention

The invention provides a method for expressing chitosanase using Rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, fermentation inoculant and applications to solve the problem that chitosanase genes in microorganisms cannot naturally Expression, the technical problem of inducing expression through chitosan and affecting the growth of microorganisms.

The technical solutions adopted by the present invention are as follows:

A method for expressing chitosanase using Rhodopseudomonas palustris. The chitosanase gene sequence encoding Bacillus subtilis is constructed into a recombinant plasmid. The recombinant plasmid is transformed into Rhodopseudomonas palustris through contact with Escherichia coli. A recombinant strain of Rhodopseudomonas palustris is obtained, and the recombinant strain of Rhodopseudomonas palustris is fermented and cultured to obtain chitosanase.

Further, the nucleotide sequence encoding the chitosanase gene of Bacillus subtilis is shown in SEQ ID NO. 1. Using the universal primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4) of the conserved sequence of the chitosanase gene, and using high-fidelity PCR technology with the genomic DNA of Bacillus subtilis CS2 (isolated and purified from soil) as a template, A chitosanase gene derived from Bacillus subtilis was cloned using primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4). Its full length is 834 bp. NCBI registration number is: OK272498. Rhodopseudomonas palustris HL-1 (isolated and purified from seawater).

Further, the fermentation culture of the recombinant strain of Rhodopseudomonas palustris includes: placing the recombinant strain of Rhodopseudomonas palustris in a fermentation medium containing 50 μg/mL of calamycin sulfate, 25-30°C, 5-25W (color temperature 3000~6500K) fluorescent lamp and anaerobic light culture for 7~10 days to obtain chitosanase. Preferably, culture at 28°C, 14W (color temperature 6500K) fluorescent lamp, and anaerobic light for 7 days. Fermentation medium includes: NaAC 0.5g/L～2g/L, ammonium oxalate 0.5g/L～2g/L, yeastextract 0.5g/L～4g/L, FeSO ₄ ·7H ₂ O 0.0005g/L～0.05g/ L, Na ₂ MoO ₄ 0.01g/L～0.05g/L. Among them, yeast extract is yeast powder. Preferably, the fermentation medium includes: NaAC 1g/L, ammonium oxalate 0.5g/L, yeastextract 2g/L, FeSO ₄ ·7H ₂ O 0.0025g/L, Na ₂ MoO ₄ 0.015g/L.

According to another aspect of the present invention, there is also provided a chitosanase obtained by the above method, the amino acid sequence of which is shown in SEQ ID NO. 2.

Further, chitosanase belongs to the glycoside hydrolase GH46 family, its molecular weight is 31.443KD, and its enzyme activity (titer) is 51U/mL. The optimal reaction temperature of chitosanase is 60°C, and the optimal pH value is 5.5. Under these conditions, the hydrolysis efficiency of chitosan (95% deacetylation degree) is 95%.

According to another aspect of the present invention, a recombinant plasmid is also provided, including the above-mentioned chitosanase gene encoding Bacillus subtilis and the pBBR1MCS-2 plasmid. Using high-fidelity PCR technology, Bacillus subtilis genomic DNA was used as a template, and primers csnS_ORF_E (SEQ ID No. 5) and csnS_ORF_X (SEQ ID No. 6) were used to clone the chitosanase gene ORF sequence with enzyme cutting sites. Recombinant plasmid pBBR1MCS-2-csnS was constructed with pBBR1MCS-2 plasmid by enzyme digestion and ligation methods. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells by heat shock, and positive clones were screened by plasmid sequencing.

According to another aspect of the present invention, a recombinant strain of Rhodopseudomonas palustris is also provided, including the above recombinant plasmid.

Further, the preparation of the recombinant strain of Rhodopseudomonas palustris includes: extracting the recombinant plasmid pBBR1MCS-2-csnS from Escherichia coli DH5α, transforming the auxotrophic Escherichia coli WM3064 competent cells by heat shock, and adding 50 μg/mL2,6 - Positive clones were screened on LB medium plates with diaminopimelic acid, 100 μg/mL streptomycin and 50 μg/mL clarithromycin sulfate. The positive clones were cultured in LB liquid medium until OD ₆₀₀ = 0.4. 2 mL of bacterial solution was centrifuged to collect the cells. After washing twice with sterile water, the cells were suspended in 1 mL of sterile physiological saline for later use. Mix 10 μL of the WM3064 bacterial liquid containing the recombinant plasmid suspended in physiological saline with 1 mL of the Rhodopseudomonas palustris bacterial liquid, and spread 100 μL on a fermentation medium plate containing 50 μg/mL 2,6-diaminopimelic acid. Culture at 28°C for 3 to 7 days. Use sterile water to elute the co-cultured colonies into a 2mL centrifuge tube, pipet 50 μL and spread it on a fermentation medium plate containing 50 μg/mL kanamycin sulfate for 7 to 10 days, and then pick a single colony to contain the same antibiotic. In the liquid fermentation medium, the recombinant strain of Rhodopseudomonas palustris was obtained under anaerobic illumination culture at 25-30°C and 5-25W (color temperature 3000-6000K) fluorescent lamp for 7-10 days.

According to another aspect of the present invention, there is also provided a fermentation agent comprising the above-mentioned Rhodopseudomonas palustris recombinant bacteria in a liquid fermentation medium containing 50 μg/mL calamicin sulfate. Medium, 25~30℃, 5~25W (color temperature 3000~6000K) fluorescent lamp, culture under anaerobic light conditions to the logarithmic growth phase to obtain a culture; then add 1~2% chitosan solution to the culture, Continue culturing for 5 to 7 days to obtain fermentation bacteria. The hydrolysis efficiency is 75%, and the cell suspension rate reaches 91.1%.

According to another aspect of the present invention, a method for using a fermentation agent comprising a recombinant bacteria that hydrolyzes chitosan oligosaccharide is also provided. The fermentation agent is diluted 200 to 1000 times directly in the pepper seedling stage, flower bud stage and flowering stage. Spray it on peppers or water them at their roots. Use it three times every 10 days to increase the wet weight and plant height of peppers.

The invention has the following beneficial effects:

The present invention uses Rhodopseudomonas palustris to express chitosanase. The chitosanase gene sequence encoding Bacillus subtilis is used to construct a recombinant plasmid. The recombinant plasmid grows in Rhodopseudomonas palustris with the growth of bacteria. Automatic expression of chitosanase does not require the addition of additional substances for induction, centrifugation, cell disruption and other prokaryotic expression protein preparation processes, making it simple and easy to implement. The chitosanase expression method of the present invention solves the disadvantage that the chitosanase gene cannot be naturally expressed in Bacillus subtilis. By constructing a recombinant plasmid containing the chitosanase gene, and expressing it in Rhodopseudomonas palustris Natural expression does not require chitosan induction and does not affect the growth of recombinant bacteria. The expressed cellular chitosanase can continuously hydrolyze chitosan to form chitosan oligosaccharide and improve the solubility of chitosan. Fermentation bacteria containing chitosan oligosaccharide. The agent can be used directly. Chitosanase is expressed along with the growth of recombinant bacteria, continuously hydrolyzes chitosan to form chitosan oligosaccharide, reduces the flocculation effect of chitosan on the bacteria, can significantly improve the suspension rate of the bacteria in the bacterial agent, and realize the chitosan oligosaccharide. The compound use of polysaccharide and microbial liquid inoculants.

In addition to the objects, features and advantages described above, the present invention has other objects, features and advantages. The present invention will be described in further detail below with reference to the accompanying drawings.

Description of the drawings

The drawings forming a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached picture:

Figure 1 is an optimal temperature diagram for hydrolyzing chitosan by chitosanase according to a preferred embodiment of the present invention, in which Temperature represents temperature and Absorption represents absorbance value;

Figure 2 is an optimal pH diagram of chitosan hydrolyzed by chitosanase according to a preferred embodiment of the present invention, where Absorption represents the absorbance value;

Figure 3 is a diagram showing the influence of the fermentation inoculant of the preferred embodiment of the present invention on the flocculation of Rhodopseudomonas palustris;

Figure 4 is a diagram of the influence of the fermentation bacteria on the growth-promoting effect of peppers according to the preferred embodiment of the present invention, in which CK represents the blank control of deionized water, and PSB represents the positive result of 200-fold dilution of wild Rhodopseudomonas palustris fermentation broth. Control, C/1 represents the positive control of the amino oligosaccharide powder solution, P200 represents the positive control of the 200-fold dilution of the fermentation agent with empty plasmid, PK200 the 200-fold dilution of the fermentation agent, Fresh weight represents the fresh weight, and Plant height represents the strain. high.

Detailed ways

It should be noted that, as long as there is no conflict, the embodiments and features in the embodiments of this application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

Figure 1 is the optimal temperature diagram for hydrolyzing chitosan by chitosanase according to the preferred embodiment of the present invention; Figure 2 is the optimal pH diagram for hydrolyzing chitosan by chitosanase according to the preferred embodiment of the present invention; Figure 3 is the optimal temperature diagram for hydrolyzing chitosan by chitosanase according to the preferred embodiment of the present invention. Figure 4 is a diagram of the influence of the fermentation bacteria in the preferred embodiment of the present invention on the flocculation of Rhodopseudomonas palustris; Figure 4 is a diagram of the influence of the fermentation bacteria in the preferred embodiment of the present invention on the growth-promoting effect of peppers.

Example

Fermentation medium: NaAC 1g/L, ammonium oxalate 0.5g/L, yeast extract 2g/L, FeSO ₄ ·7H ₂ O 0.0025g/L, Na ₂ MoO ₄ 0.015g/L, solid medium added 2g/L agar , pH is natural, the antibiotic used is: kanamycin sulfate, the concentration is 50μg/mL.

LB medium: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, pH value 7.0. Add 2g/L agar powder to solid culture medium.

Example 1

Acquisition of chitosanase gene

Align the known chitosanase gene sequences, and design the universal primers _F (SEQ ID No. 3) and _R (SEQ ID No. 4) of the gene based on the compared conserved sequences.

Genomic DNA of Bacillus subtilis CS2 was extracted by CTAB method. Use conserved sequence primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4), and use Bacillus subtilis genomic DNA as a template to amplify the complete ORF fragment of the target gene. The total volume of the PCR reaction is 50 μL: 10×Top Taq buffer 5 μL, genomic DNA template 1 μL, dNTPs 4 μL, csnS_F primer 1 μL, csnS_R primer 1 μL, Top Taq DNA polymerase 1 μL, ddH ₂ O 37 μL. The PCR reaction program is: pre-denaturation at 95°C for 5 min, then denaturation at 94°C for 30 sec, annealing at 53°C for 30 sec, extension at 72°C for 30 sec, 30 cycles, and finally final extension at 72°C for 2 min.

The PCR amplification product was purified and recovered and connected to a T vector for sequencing. After sequence comparison analysis, the complete chitosanase gene was obtained. Its nucleotide sequence is shown in SEQ ID No. 1, with a full length of 834 bp. NCBI accession number: OK272498.

Example 2

Construction of recombinant plasmid pBBR1MCS-2-csnS

Using csnS_ORF_E (SEQ ID No. 5) and csnS_ORF_X (SEQ ID No. 6) primers, under the same PCR conditions as in Example 1, a complete ORF fragment with EcoRI and XholI restriction enzyme sites was prepared. The pBBR1MCS-2 plasmid and gene ORF fragment were digested with EcoRⅠ and Positive clones were screened by culturing on LB plates containing mL of kanamycin sulfate. The positive clone was confirmed by sequencing and comparison, and the recombinant plasmid pBBR1MCS-2-csnS was obtained.

Example 3

Preparation of recombinant bacteria of Rhodopseudomonas palustris

The recombinant plasmid pBBR1MCS-2-csnS was extracted from Escherichia coli DH5α, transformed into auxotrophic Escherichia coli WM3064 competent cells by heat shock, and added with 50 μg/mL 2,6-diaminopimelic acid, 100 μg/mL streptomycin and Positive clones were screened on LB medium plates containing 50 μg/mL clarithromycin sulfate. The positive clones were cultured in LB liquid medium until OD ₆₀₀ = 0.4. 2 mL of bacterial solution was centrifuged to collect the cells. After washing twice with sterile water, the cells were suspended in 1 mL of sterile physiological saline for later use.

Mix 10 μL of the WM3064 bacterial liquid containing the recombinant plasmid suspended in physiological saline with 1 mL of Rhodopseudomonas palustris bacteria liquid, and spread 100 μL on a fermentation medium plate containing 50 μg/mL 2,6-diaminopimelic acid. , cultured at 28°C for 3 days. Use sterile water to elute the co-cultured colonies into a 2 mL centrifuge tube, pipet 50 μL and spread it on a fermentation medium plate containing 50 μg/mL calithromycin sulfate for 10 days. Pick a single colony and transfer it to a liquid fermentation culture containing the same antibiotic. The recombinant strain of Rhodopseudomonas palustris was obtained by culturing under anaerobic light under 28°C and 14W/6500K fluorescent lamp for 7 days.

Example 4

Expression and extraction of chitosanase

The recombinant strain of Rhodopseudomonas palustris was cultured in a fermentation medium containing 50 μg/mL kanamycin sulfate at 28°C, 14W/6500K fluorescent lamp, and anaerobic light for 7 days. Collect the fermentation broth supernatant by centrifugation to obtain chitosanase.

Chitosanase belongs to the glycoside hydrolase GH46 family, its molecular weight is 31.443KD, and its enzyme activity (titer) is 51U/mL.

Example 5

Biochemical properties of chitosanase

(1)Reaction temperature of chitosanase

Take a certain amount of the chitosanase extracted in Example 4, and measure the hydrolysis of 1g/L chitosan per unit time by the same volume of chitosanase in a temperature gradient water bath from 30°C to 80°C (interval 10°C). The yield of chitosan oligosaccharide generated from sugar is determined based on the content of chitosan oligosaccharide generated to determine the hydrolysis temperature curve and optimal hydrolysis temperature of the enzyme.

(2)Reaction pH of chitosanase

Take a certain amount of the chitosanase extracted in Example 4, react in an acetic acid-sodium acetate buffer with a pH value of 4.5 to 7.0 (pH interval 0.5 unit), and measure the same volume of chitosan under different pH conditions. Carbohydrase hydrolyzes 1g/L chitosan to produce chitosan oligosaccharide in unit time to determine the hydrolysis pH curve and optimal pH of the enzyme.

As shown in Figure 1, the reaction temperature of chitosanase is 30°C to 65°C, and the optimal reaction temperature is 60°C.

As shown in Figure 2, the reaction pH value of chitosanase is 4.5-6, and the optimal reaction pH value is 5.5.

Example 6

Preparation of fermentation agent of Rhodopseudomonas palustris recombinant bacteria and detection of cell suspension rate

The recombinant Rhodopseudomonas palustris in Example 3 was cultured to the mid-logarithmic growth stage in a liquid fermentation medium containing 50 μg/mL calithromycin sulfate under anaerobic light conditions at 28°C, 14W/6500K fluorescent lamp, Then add 1% chitosan solution to the culture and continue culturing for 7 days to obtain the fermentation agent.

Shake and suspend the fermentation bacteria thoroughly, quickly and accurately weigh 50g (about 50mL) sample to the nearest 0.0001g, place it in a 200mL beaker containing 50mL sterile deionized water (25±1℃), and shake it by hand Make circular motions, about 120 times per minute, for 2 minutes. Place the suspension in a water bath at the same temperature for 13 minutes, then wash it all into a 250mL graduated cylinder with sterile deionized water (25±1°C), dilute to the mark, cover with a stopper, and use the bottom of the graduated cylinder as the axis. , turn the measuring cylinder upside down 30 times within 1 minute (turn the measuring cylinder upside down and return it to its original position once, about 2 seconds). Open the stopper and place it vertically into a vibration-free constant temperature water bath for 30 minutes. Use a pipette to remove 9/10 (i.e. 225mL) of the suspension of the contents within 10 to 15 seconds. Do not shake or stir up the sediment in the measuring cylinder. Make sure that the top of the pipette is always a few millimeters below the liquid surface.

Determination of the bacterial content of the sample and the 25 mL suspension left at the bottom of the measuring cylinder: 25°C, 10,000 rpm, centrifuge for 10 minutes, suck out the supernatant, invert the centrifuge tube and blow dry the water, and then weigh the weight of the bacterial cells.

Result calculation:

In the formula, m ₁ is the mass of bacteria contained in the sample taken to prepare the suspension, in g; m ₂ is the mass of bacteria contained in the 25 mL suspension left at the bottom of the graduated cylinder, in g.

As shown in Figure 3, the right side is the fermentation agent of the recombinant strain of Rhodopseudomonas palustris, and the left side is the control group. The suspension rate of the fermentation agent of the recombinant Rhodopseudomonas palustris reached 91.1%, while the Rhodopseudomonas palustris control group (CK) containing only the original plasmid pBBR1MCS-2 showed a large amount of precipitation, and the suspension rate of the bacteria was 9.2%. .

Example 7

The growth-promoting effect of fermentation bacteria on pepper

The fermentation agent of Example 6 was diluted 200, 400, 600, 800, and 1000 times in sequence, and deionized water was used as a blank control. The fermentation broth of wild Rhodopseudomonas palustris was diluted 200 times, 0.05g/L commercial product The amino oligosaccharide powder solution and the 200-fold dilution of the fermentation agent with empty plasmid were used as positive controls, and the pepper seedlings were sprayed respectively. Each treatment group contains 5 pepper seedlings with 4 true leaves. They are sprayed for the first time 10 days after transplanting, and then sprayed once every 10 days, for a total of 3 sprays. Each one is counted on the 15th day after the third spraying. Fresh weight and plant height of treatment groups.

The fermentation inoculant of the present invention can obviously promote the growth of peppers, and the fresh weight and plant height of peppers will be increased after being treated with different dilutions of the fermentation inoculants. As shown in Figure 4, the dilution of fermentation agent 200 was significantly higher than that of the blank control and the positive control. Among them, the fresh weight of peppers treated with the dilution of fermentation agent 200 increased by 16.6% compared to the blank control. The height increased by 13.9%, which significantly promoted the growth of peppers.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

sequence list

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Claims (2)

1. A fermentation agent comprising a recombinant strain of Rhodopseudomonas palustris, characterized by: The Rhodopseudomonas palustris recombinant bacterium includes a recombinant plasmid, the recombinant plasmid includes a chitosanase gene encoding Bacillus subtilis and a pBBR1MCS-2 plasmid, and the nucleoside encoding the chitosanase gene of Bacillus subtilis The acid sequence is shown in SEQ ID NO.1; The preparation of the Rhodopseudomonas palustris recombinant bacteria includes the following steps: The recombinant plasmid is transformed into Escherichia coli WM3064 by heat shock. The positive transformant is co-cultured with Rhodopseudomonas palustris for 3 to 7 days. The co-cultured bacteria are eluted from the culture medium plate and then spread onto the culture medium containing clarithromycin sulfate. On the base plate, screen the recombinant bacteria of Rhodopseudomonas palustris; The preparation of the fermentation inoculant includes the following steps: The recombinant strain of Rhodopseudomonas palustris was cultured under anaerobic light conditions in a liquid fermentation medium containing 50 μg/mL calamycin sulfate under 25-30°C, 5-25W (color temperature 3000-6500K) fluorescent lamps. After several growth periods, the culture is obtained, and then 1 to 2% chitosan solution is added to the culture, and the culture is continued for 5 to 7 days to obtain the fermentation agent. 2. The application of the fermentation inoculant according to claim 1 in pepper growth promotion, characterized in that, The fermentation inoculant is diluted 200 to 1000 times and then sprayed at the pepper seedling stage, flower bud stage and flowering stage, or used for root irrigation.