CN113913450B

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

Info

Publication number: CN113913450B
Application number: CN202111268892.XA
Authority: CN
Inventors: 王忠勇; 刘勇; 张德咏; 张宇; 卢丁伊慧
Original assignee: HUNAN PLANT PROTECTION INSTITUTE
Current assignee: HUNAN PLANT PROTECTION INSTITUTE
Priority date: 2021-10-29
Filing date: 2021-10-29
Publication date: 2024-02-02
Anticipated expiration: 2041-10-29
Also published as: CN113913450A

Abstract

The invention discloses a method for expressing chitosanase by utilizing rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, zymophyte and application. According to the method for expressing the chitosanase by utilizing rhodopseudomonas palustris, the recombinant plasmid containing the chitosanase gene is constructed and naturally expressed in rhodopseudomonas palustris, chitosan induction is not needed, the growth of recombinant bacteria is not influenced, the expressed chitosanase can continuously hydrolyze chitosan to form chitosan oligosaccharide, the dissolubility of chitosan is improved, and the zymophyte containing the chitosan oligosaccharide can be directly used.

Description

Method for expressing chitosanase by rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, fermentation bacteria and application

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for expressing chitosanase by utilizing rhodopseudomonas palustris. In addition, the invention also relates to chitosanase, recombinant plasmid, recombinant bacteria, zymophyte and application thereof.

Background

Chitosan (chitosan) is a product obtained by removing part of N-deacetylation from chitin, and has many characteristics and is widely applied to the agricultural field. However, chitosan with high polymerization degree has large molecular weight and poor water solubility, is not easy to be absorbed by plants, has extremely strong flocculation effect, and severely limits the direct use of chitosan and the compound use of chitosan with other biological agents and microbial agents. The chitosan is hydrolyzed by chitosan enzyme (EC 3.2.1.132), so that the polymerization degree of the chitosan is reduced, chitosan oligosaccharide is produced, and various defects of the chitosan in use are greatly overcome.

At present, the chitosan enzyme genes are mainly from bacteria and fungus microorganisms, but the chitosan enzyme genes in the microorganisms cannot be expressed naturally, and the chitosan needs to be added into a culture medium to induce the gene expression. However, chitosan is insoluble in water, and needs to be dissolved by acetic acid or even hydrochloric acid, and the chitosan colloid solution seriously influences the pH of the culture medium after being added into the culture medium, thereby influencing the growth of microorganisms. Secondly, the chitosan colloid solution has very poor stability, is easy to separate out after being added into a culture medium, has very strong flocculation effect on microorganisms, and further limits the growth of the microorganisms, so that the chitosan is not expressed or expressed in a small amount.

Disclosure of Invention

The invention provides a method for expressing chitosanase by utilizing rhodopseudomonas palustris, chitosanase, recombinant plasmid, recombinant bacteria, zymophyte agent and application thereof, which are used for solving the technical problems that chitosanase genes in microorganisms cannot be expressed naturally and the growth of the microorganisms is influenced by the induction and expression of chitosan.

The technical scheme adopted by the invention is as follows:

a method for expressing chitosanase by utilizing rhodopseudomonas palustris comprises the steps of constructing recombinant plasmid by a chitosanase gene sequence for encoding bacillus subtilis, converting the recombinant plasmid into rhodopseudomonas palustris in a body by escherichia coli contact to obtain rhodopseudomonas palustris recombinant strain, and fermenting and culturing the rhodopseudomonas palustris recombinant strain to obtain chitosanase.

Further, the nucleotide sequence of the chitosan enzyme gene of the encoding bacillus subtilis is shown as SEQ ID NO. 1. Using the general primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4) of the conserved sequence of the chitosanase gene, a chitosanase gene derived from bacillus subtilis CS2 (from soil isolation and purification) was cloned by high-fidelity PCR technique using the primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4) as templates, the total length of which was 834bp. NCBI accession number is: OK272498. Rhodopseudomonas palustris HL-1 (from seawater separation and purification).

Further, the rhodopseudomonas palustris recombinant strain fermentation culture comprises the following steps: the rhodopseudomonas palustris recombinant is cultured in a fermentation medium containing 50 mug/mL of caliamycin sulfate for 7-10 days by anaerobic illumination at 25-30 ℃ under a fluorescent lamp with a color temperature of 5-25W (3000-6500K). Preferably, a fluorescent lamp at 28℃and 14W (color temperature 6500K) is used for 7 days of anaerobic light cultivation. The fermentation medium comprises: naAC 0.5 g/L-2 g/L, ammonium oxalate 0.5 g/L-2 g/L, dye extract0.5 g/L-4 g/L, feSO ₄ ·7H ₂ O 0.0005g/L～0.05g/L，Na ₂ MoO ₄ 0.01g/L to 0.05g/L. Wherein the yeast extract is yeast powder. Preferably, the fermentation medium comprises: naAC 1g/L, ammonium oxalate 0.5g/L, eye extract 2g/L, feSO ₄ ·7H ₂ O0.0025g/L，Na ₂ MoO ₄ 0.015g/L。

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

Further, the chitosanase belongs to the glycoside hydrolase GH46 family, and has a molecular weight of 31.443KD and an enzyme activity (potency) of 51U/mL. The optimum reaction temperature of the chitosanase is 60 ℃, the optimum pH value is 5.5, and the hydrolysis efficiency of chitosan (95% deacetylation degree) under the condition is 95%.

According to another aspect of the present invention, there is also provided a recombinant plasmid comprising the above-mentioned chitosanase gene encoding Bacillus subtilis and a pBBR1MCS-2 plasmid. The recombinant plasmid pBBR1MCS-2-csnS is constructed by using the high-fidelity PCR technology and using bacillus subtilis genome DNA as a template, cloning a chitosan enzyme gene ORF sequence with an enzyme cutting site by using primers csnS_ORF_E (SEQ ID No. 5) and csnS_ORF_X (SEQ ID No. 6) and constructing the recombinant plasmid pBBR1MCS-2-csnS with a pBBR1MCS-2 plasmid by an enzyme cutting and connecting method. E.coli DH5 alpha competent cells were transformed by heat shock of the recombinant plasmid, and positive clones were screened by plasmid sequencing.

According to another aspect of the invention, there is also provided a rhodopseudomonas palustris recombinant bacterium comprising the recombinant plasmid.

Further, the preparation of rhodopseudomonas palustris recombinant bacteria comprises the following steps: recombinant plasmid pBBR1MCS-2-csnS was extracted from E.coli DH 5. Alpha. And auxotrophic E.coli WM3064 competent cells were transformed by heat shock, and positive clones were selected on LB medium plates supplemented with 50. Mu.g/mL 2, 6-diaminopimelic acid, 100. Mu.g/mL streptomycin and 50. Mu.g/mL calicheamicin sulfate. Positive clones were cultured in LB liquid medium to OD ₆₀₀ 2mL of bacterial liquid was centrifuged to collect the cells, which were washed twice with sterile water and suspended in 1mL of sterile physiological saline for use. After 10. Mu.L of a physiological saline suspension WM3064 bacterial liquid containing recombinant plasmid and 1mL of rhodopseudomonas palustris bacterial liquid are mixed, 100. Mu.L of the mixture is coated on a fermentation medium flat plate containing 50. Mu.g/mL 2, 6-diaminopimelic acid, and the mixture is co-cultured for 3 to 7 days at 28 ℃. The co-cultured colony is eluted by using sterile water and placed in a 2mL centrifuge tube, 50 mu L of the co-cultured colony is absorbed and coated on a fermentation culture medium flat plate containing 50 mu g/mL kanamycin sulfate for 7-10 days, then a single colony is picked up and placed in a liquid fermentation culture medium containing the same antibiotics for 7-10 days by using a fluorescent lamp with the temperature of 25-30 ℃ and the color temperature of 5-25W (3000-6000K) for anaerobic illumination, and the rhodopseudomonas palustris recombinant strain is obtained.

According to another aspect of the present invention, there is also provided a fermentation inoculant comprising the recombinant rhodopseudomonas palustris as described above, the recombinant rhodopseudomonas palustris being cultivated in a liquid fermentation medium containing 50 μg/mL of caliamycin sulfate at 25-30 ℃, a fluorescent lamp at 5-25W (color temperature 3000-6000K) under anaerobic illumination conditions to a logarithmic growth phase, to obtain a culture; and adding 1-2% chitosan solution into the culture, and continuously culturing for 5-7 days to obtain the fermentation inoculum. The hydrolysis efficiency is 75%, and the thallus suspension rate reaches 91.1%.

According to the application method of the fermentation inoculant of the recombinant bacteria comprising the hydrolyzed chitosan oligosaccharide, the fermentation inoculant is directly sprayed on the peppers in the seedling stage, the bud stage and the flower stage of the peppers by 200-1000 times of diluent, or irrigated on the roots of the peppers, and the fermentation inoculant is continuously used for 3 times every 10 days, so that the wet weight and the plant height of the peppers can be improved.

The invention has the following beneficial effects:

according to the method for expressing the chitosanase by using rhodopseudomonas palustris, the gene sequence of the chitosanase for encoding the bacillus subtilis is used for constructing the recombinant plasmid, the recombinant plasmid automatically expresses the chitosanase along with the growth of thalli in rhodopseudomonas palustris, and the preparation process of prokaryotic expression proteins such as substance induction, centrifugation, thalli crushing and the like is not needed, so that the method is simple and easy to implement. The expression method of chitosan enzyme solves the defect that the chitosan enzyme gene cannot be expressed naturally in bacillus subtilis, and the recombinant plasmid containing the chitosan enzyme gene is constructed and expressed naturally in rhodopseudomonas palustris without chitosan induction and influence on the growth of recombinant bacteria, so that the expressed cytoskeletal glycanase can continuously hydrolyze chitosan to form chitosan oligosaccharide, the solubility of chitosan is improved, and the fermentation inoculant containing chitosan oligosaccharide can be directly used. The chitosan enzyme is expressed along with the growth of recombinant bacteria, and continuously hydrolyzes chitosan to form chitosan oligosaccharide, so that the flocculation effect of chitosan on the bacteria is reduced, the suspension rate of the bacteria in the microbial agent can be obviously improved, and the compound use of chitosan and microbial liquid microbial agent is realized.

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

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is an optimum Temperature diagram of chitosan hydrolyzed by chitosan enzyme according to a preferred embodiment of the present invention, wherein Temperature represents Temperature and absorbance represents absorbance value;

FIG. 2 is a graph of the pH optimum for chitosan hydrolysis by a chitosan enzyme according to a preferred embodiment of the present invention, wherein absorptions represent absorbance values;

FIG. 3 is a graph showing the effect of a fermenting agent according to a preferred embodiment of the present invention on flocculation of rhodopseudomonas palustris;

FIG. 4 is a graph showing the effect of the fermentation broth of the preferred embodiment of the present invention on the growth promoting effect of capsicum, wherein CK represents a control blank of deionized water, PSB represents a positive control of 200-fold dilutions of the fermentation broth of P.palustris, C/1 represents a positive control of a solution of amino-oligosaccharide powder, P200 represents a positive control of 200-fold dilutions of the fermentation broth of empty plasmid, PK200 represents 200-fold dilutions of the fermentation broth, fresh weight represents Fresh weight, and Plant height represents Plant height.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

FIG. 1 is an optimum temperature diagram of chitosan hydrolyzed by chitosan enzyme according to a preferred embodiment of the present invention; FIG. 2 is a graph showing the optimum pH for chitosan hydrolysis by the chitosanase of the preferred embodiment of the present invention; FIG. 3 is a graph showing the effect of a fermenting agent according to a preferred embodiment of the present invention on flocculation of rhodopseudomonas palustris; FIG. 4 is a graph showing the effect of the fermentation broth of the preferred embodiment of the present invention on the growth promoting effect of capsicum.

Examples

Fermentation medium: naAC 1g/L, ammonium oxalate 0.5g/L, eye extract 2g/L, feSO ₄ ·7H ₂ O 0.0025g/L，Na ₂ MoO ₄ 0.015g/L, 2g/L agar is added into a solid culture medium, the pH is natural, and the antibiotics are: kanamycin sulfate at a concentration of 50 μg/mL.

LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride and pH 7.0. 2g/L of agar powder is added into the solid culture medium.

Example 1

Acquisition of chitosanase Gene

The sequences of known chitosanase genes are aligned, and the general primers of the genes, namely, the_F (SEQ ID No. 3) and the_R (SEQ ID No. 4), are designed according to the conserved sequences after alignment.

The CTAB method is used for extracting the genome DNA of the bacillus subtilis CS 2. The complete ORF fragment of the gene of interest was amplified using the conserved sequence primers csnS_F (SEQ ID No. 3) and csnS_R (SEQ ID No. 4) and using the Bacillus subtilis genomic DNA as template. The total volume of PCR reaction was 50. Mu.L: 10 xTop Taq buffer 5. Mu.L, genomic DNA template 1. Mu.L, dNTPs 4. Mu.L, csnS_F primer 1. Mu.L, csnS_R primer 1. Mu.L, top Taq DNA polymerase. Mu.L, ddH ₂ O37. Mu.L. The PCR reaction procedure was: pre-denaturation at 95℃for 5min, then denaturation at 94℃for 30sec, annealing at 53℃for 30sec, elongation at 72℃for 30sec,30 cycles, and final elongation at 72℃for 2min.

And (3) after purifying and recovering the PCR amplification product, sequencing the ligation T vector, and comparing and analyzing the sequences to obtain the complete chitosan enzyme gene, wherein the nucleotide sequence of the complete chitosan enzyme gene is shown as SEQ ID No.1, and the total length of the complete chitosan enzyme gene is 834bp. NCBI accession number: OK272498.

Example 2

Construction of recombinant plasmid pBBR1MCS-2-csnS

Complete ORF fragments with EcoRI and XholI restriction sites were prepared using the 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. The pBBR1MCS-2 plasmid and the gene ORF fragment were digested with EcoRI and XholI restriction enzymes, respectively, and after agarose gel purification and recovery, E.coli DH 5. Alpha. Competent cells were transformed by heat shock using the ligation product, and positive clones were screened by culturing on LB plates supplemented with 50. Mu.g/mL kanamycin sulfate. The positive clone is subjected to sequencing comparison and confirmation to obtain the recombinant plasmid pBBR1MCS-2-csnS.

Example 3

Preparation of rhodopseudomonas palustris recombinant bacteria

Recombinant plasmid pBBR1MCS-2-csnS was extracted from E.coli DH 5. Alpha. And auxotrophic E.coli WM3064 competent cells were transformed by heat shock, and positive clones were selected on LB medium plates supplemented with 50. Mu.g/mL 2, 6-diaminopimelic acid, 100. Mu.g/mL streptomycin and 50. Mu.g/mL calicheamicin sulfate. Positive clones were cultured in LB liquid medium to OD ₆₀₀ 2mL of bacterial liquid was centrifuged to collect the cells, which were washed twice with sterile water and suspended in 1mL of sterile physiological saline for use.

After 10. Mu.L of a physiological saline-suspended WM3064 bacterial solution containing the recombinant plasmid was mixed with 1mL of Rhodopseudomonas palustris bacterial solution, 100. Mu.L was spread on a fermentation medium plate containing 50. Mu.g/mL 2, 6-diaminopimelic acid, and co-cultured at 28℃for 3 days. The co-cultured colony is eluted by using sterile water and placed in a 2mL centrifuge tube, 50 mu L of the co-cultured colony is absorbed and coated on a fermentation medium flat plate containing 50 mu g/mL of caliamycin sulfate for 10 days, a single colony is picked up and placed in a liquid fermentation medium containing the same antibiotics, and anaerobic illumination culture is carried out for 7 days by using a 14W/6500K fluorescent lamp at the temperature of 28 ℃ to obtain the rhodopseudomonas palustris recombinant strain.

Example 4

Expression and extraction of chitosanase

Rhodopseudomonas palustris recombinant strain is cultured in fermentation medium containing 50 μg/mL kanamycin sulfate at 28deg.C under anaerobic illumination for 7 days under 14W/6500K fluorescent lamp. And centrifuging to collect supernatant of the fermentation broth to obtain the chitosanase.

Chitosanase belongs to the family of glycoside hydrolase GH46, and has a molecular weight of 31.443KD and an enzyme activity (potency) of 51U/mL.

Example 5

Biochemical Properties of chitosanase

(1) Reaction temperature of chitosanase

Taking a certain amount of chitosanase extracted in the example 4, respectively in a temperature gradient water bath with a temperature of 30 ℃ to 80 ℃ (with an interval of 10 ℃), measuring the yield of chitosan oligosaccharide generated by hydrolyzing 1g/L chitosan with the same volume of chitosanase in unit time, and determining the hydrolysis temperature curve and the optimal hydrolysis temperature of the enzyme according to the content of the generated chitooligosaccharide.

(2) Reaction pH of chitosanase

Taking a certain amount of chitosan enzyme extracted in the example 4, respectively carrying out reaction in acetic acid-sodium acetate buffer solution with the pH value of 4.5-7.0 (the pH interval is 0.5 unit), and measuring the yield of chitosan oligosaccharide generated by hydrolyzing 1g/L chitosan by the same volume of chitosan enzyme under different pH conditions in unit time so as to determine the hydrolysis pH curve and the optimal pH of the enzyme.

As shown in FIG. 1, the reaction temperature of the chitosanase is 30-65 ℃, and the optimal reaction temperature is 60 ℃.

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

Example 6

Preparation of fermenting agent of rhodopseudomonas palustris recombinant bacteria and detection of thallus suspension rate

The recombinant rhodopseudomonas palustris of example 3 is cultivated in a liquid fermentation medium containing 50 mug/mL of caliamycin sulfate at 28 ℃ under 14W/6500K fluorescent lamp and anaerobic illumination condition until the mid-log growth period, and then 1% chitosan solution is added into the culture, and the cultivation is continued for 7 days to obtain the fermentation inoculum.

The zymophyte agent is fully and evenly shaken for suspension, 50g (about 50 mL) of sample is rapidly and accurately weighed to 0.0001g, and the sample is placed in a 200mL beaker containing 50mL of sterile deionized water (25+/-1 ℃) and is oscillated by hand for circular motion, about 120 times per minute for 2min. The suspension was placed in a water bath at the same temperature for 13min, then washed with sterile deionized water (25.+ -. 1 ℃) all into a 250mL graduated cylinder and diluted to the scale, capped with a stopper, and the cylinder was turned upside down 30 times (the cylinder was turned upside down and returned to its original position once for about 2 s) within 1 min. The plug was opened and then placed vertically in a vibration-free thermostatic water bath for 30min. The 9/10 (i.e., 225 mL) suspension of the contents was removed with the pipette over 10-15 seconds without shaking or stirring the sediment in the cylinder, ensuring that the tip of the pipette was always a few millimeters below the liquid surface.

Determination of the sample and the cell content in 25mL of suspension left in the bottom of the measuring cylinder: centrifuging at 25deg.C and 10000rpm for 10min, sucking out supernatant, drying the water in the inverted centrifuge tube, and weighing the thallus respectively.

And (3) calculating results:

wherein m is ₁ The mass of thalli contained in a sample for preparing suspension is in g; m is m ₂ The mass of the cells contained in 25mL of the suspension remaining in the bottom of the measuring cylinder is expressed in g.

As shown in FIG. 3, the right side is the fermentation inoculum of rhodopseudomonas palustris recombinant bacteria, and the left side is the control group. The suspension rate of the fermentation inoculum of the rhodopseudomonas palustris recombinant bacteria reaches 91.1%, while the rhodopseudomonas palustris control group (CK) only containing pBBR1MCS-2 original plasmid has a large amount of precipitation, and the thallus suspension rate is 9.2%.

Example 7

Growth promoting effect of fermentation inoculant on capsicum

The fermentation inoculant of example 6 was diluted 200, 400, 600, 800 and 1000 times in sequence, deionized water was used as a blank control, 200 times of the dilution of the fermentation broth of the wild rhodopseudomonas palustris, 0.05g/L of the commercial amino-oligosaccharide powder solution and 200 times of the dilution of the fermentation inoculant with empty plasmids were used as positive controls, and pepper seedlings were sprayed respectively. Each treatment group comprises 5 plants of 4 pieces of real leaf pepper seedlings, the first spraying is carried out after 10 days of transplanting, the spraying is carried out once every 10 days, the total spraying is carried out for 3 times, and the fresh weight and the plant height of each treatment group are counted on 15 days after the third spraying.

The fermentation inoculant disclosed by the invention obviously promotes the growth of the peppers, and the fresh weight and the plant height of the peppers can be improved after the peppers are treated by different dilutions of the fermentation inoculant. As shown in fig. 4, the diluted solution of the zymophyte agent 200 is significantly higher than that of the blank control and the positive control, wherein the fresh weight of the capsicum treated by the diluted solution of the zymophyte agent 200 is improved by 16.6% and the plant height is improved by 13.9% compared with the blank control, namely the growth of the capsicum is significantly promoted.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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Claims

1. A zymophyte agent comprising rhodopseudomonas palustris recombinant bacteria is characterized in that,

the rhodopseudomonas palustris recombinant bacteria comprise recombinant plasmids, wherein the recombinant plasmids comprise a chitosan enzyme gene for encoding bacillus subtilis and a pBBR1MCS-2 plasmid, and the nucleotide sequence of the chitosan enzyme gene for encoding the bacillus subtilis is shown as SEQ ID NO. 1;

the preparation method of the rhodopseudomonas palustris recombinant strain comprises the following steps:

the recombinant plasmid is subjected to heat shock conversion to escherichia coli WM3064, the positive transformant and rhodopseudomonas palustris are co-cultured for 3 to 7 days, the co-cultured bacteria are eluted from a culture medium plate and then coated on a culture medium plate containing caliamycin sulfate, and rhodopseudomonas palustris recombinant bacteria are screened;

the preparation of the fermentation inoculant comprises the following steps:

the rhodopseudomonas palustris recombinant is cultivated in a liquid fermentation medium containing 50 mug/mL of caliamycin sulfate at 25-30 ℃ and a fluorescent lamp at 5-25W (with a color temperature of 3000-6500K) under the anaerobic illumination condition until the bacterial strain is in a logarithmic growth phase to obtain a culture, and then 1-2% of chitosan solution is added into the culture to continue to cultivate for 5-7 days to obtain the fermentation inoculum.

2. A method for promoting the growth of capsicum using the fermenting bacteria agent of claim 1,

the fermentation inoculant is diluted by 200-1000 times and then sprayed in the seedling stage, the bud stage and the flowering stage of the capsicum, or used for root irrigation.