CN118546819A - Construction and application of bacillus tropicalis chitosan enzyme BtCSN surface display method - Google Patents

Abstract

The present invention relates to the field of bioengineering technology, and specifically discloses the construction and application of a tropical bacillus chitosanase BtCSN surface display method. The present invention provides a tropical bacillus, which is tropical bacillus CSN-15, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on May 23, 2022, and the deposit number is: CGMCC No.24948. The strain can produce chitosanase BtCSN with higher activity. The present invention is based on pINA1317-YlCWP110-lipolytica Yarrowia lipolytica surface display technology system, and displays tropical bacillus chitosanase BtCSN on the surface of Yarrowia lipolytica yeast cells. The activity of the surface display transformant is as high as 2836.66±16.21U/ml cell suspension, and its whole cell catalysis can be reused 6 times and still maintain about 70% of the activity, which has good application prospects. And preliminarily establish the production process conditions for preparing chitosan oligosaccharides by whole cell catalysis, laying a solid foundation for its large-scale preparation of chitosan oligosaccharides.

Description

Construction and application of a surface display method for Bacillus tropicalis chitosanase BtCSN

Technical Field

The invention relates to the technical field of bioengineering, and in particular to the construction and application of a surface display method of tropical bacillus chitosanase BtCSN.

Background Art

Chitosan is the only natural alkaline polysaccharide, but its development and utilization are severely limited due to its large molecular weight and poor solubility in water. 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. Chitosanase is a glycoside hydrolase that can specifically degrade chitosan. The preparation of chitosan oligosaccharides by microbial enzymatic degradation of chitosan has the advantages of mild conditions, controllable products, and environmental friendliness. In recent years, the use of specific chitosanase to hydrolyze chitosan to prepare chitosan oligosaccharides has become a research hotspot and frontier in the chitosan industry.

However, during the catalytic process of chitosanase, there are problems such as severe loss of enzyme activity, inability to continuously catalyze, and inability to recycle free enzymes, which leads to high production costs and waste of enzyme resources. At present, chitosanase research is mostly focused on strain screening and free cell enzyme production conditions. Therefore, the immobilization technology of surface display cells is of great significance for improving the production and application efficiency of chitosanase. Therefore, a chitosanase BtCSN surface display method is provided to overcome the various limitations of chitosanase in industrial applications, reduce production costs, and realize industrial biocatalysis and biosynthesis pathways based on green synthases.

Summary of the invention

The purpose of the present invention is to address the defects of the prior art and to provide a method for constructing and applying a surface display method of tropical Bacillus subtilis chitosanase BtCSN to solve the problems raised by the above-mentioned background technology.

To achieve the above-mentioned purpose, the present invention provides the following technical solution: a tropical Bacillus, namely tropical Bacillus CSN-15, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on May 23, 2022, with the deposit number: CGMCC No.24948, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Bacillus tropicus.

The invention discloses an application of tropical bacillus in producing chitosanase BtCSN with high activity, characterized in that the specific production steps are as follows:

S1: Activation of strains: Bacillus tropicus CSN-15 stored at -80°C was activated on LB medium plates at 37°C for 18 h;

S2: Preparation of Bacillus tropicus chitosanase BtCSN fragment: Using Bacillus tropicusCSN-15 as template, use BtCSN-F/R primer pair to amplify the full length of BtCSN gene. The PCR system and procedure are as follows:

Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 5 min;

S3: The recovered gene fragments were connected to the pMD19-T simple vector using the T vector ligation kit TaKaRa pMD19-T simple Vector to construct pMD19-T simple-BtCSN. The system and conditions are as follows:

Connect at 16℃ for 16h;

S4: Transformation of recombinant plasmid into E. coli DH5α;

S41: Take 50.0 μl of DH5α competent cell suspension from the -80°C refrigerator and place on ice to thaw;

S42: Add the DNA solution to be transformed, the content of the DNA solution should not exceed 50 ng, the volume should not exceed 10 μl, gently rotate to mix, and place on ice for 30 min;

S43: Place in a 42°C water bath and heat shock for 90 seconds. Do not shake the tube during this period.

S44: Quickly transfer the tube to ice and allow the cells to cool for 1-2 minutes;

S45: Add 950 μl LB liquid medium, mix well, and culture on a shaker at 37°C and 180 rpm for 45 min to allow the cells to recover and express the Amp resistance gene encoded by the plasmid;

S46: Centrifuge the cultured bacterial solution at 5000×g for 5 min, remove 600 μl of the supernatant, and then resuspend the cells. Take 100.0 μl of the cell suspension and apply it on a LA screening plate containing 40.0 μl of 20 mg/ml X-gal solution and 7.0 μl of 20% IPTG solution;

S47: Place the plate upright in a 37°C incubator and culture for 30 minutes. After the bacterial solution is absorbed, invert the plate and continue culturing. Colonies can be observed after 12-16 hours.

As a preferred technical solution of the present invention, the LB medium in step S1: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, 2% agar powder, sterilized at 120°C for 20 minutes, and then cooled to room temperature.

As a preferred technical solution of the present invention, the BtCSN-F primer sequence in step S2 is: AGGCCGTTCTGGCCTACAACCTTCCAAACAACCTCAAG;

Primer sequence for BtCSN-R: CGCGGATCCTGCCTTCAGACCTGCGACCAGCTT; restriction enzyme sites at both ends are Sfi I and BamHI respectively.

A method for surface display of chitosanase BtCSN is constructed. Based on the surface display system of Yarrowia lipolytica, chitosanase BtCSN is recombined into the surface display plasmid pINA1317-YICWP110 to construct the recombinant plasmid pINA1317-YICWP110-BtCSN.

The specific steps are as follows:

Step 1: Double digest the plasmids pMD19-T simple-BtCSN and pINA1317-YlCWP110 with SfiI and BamHI, respectively. The digestion conditions and system are as follows:

50℃, overnight digestion;

Then, 2.0 μl BamHI was added and digested at 37°C for 8 h. After the digestion was completed, 5.0 μl of the digestion product was taken and subjected to 1.0% agarose gel electrophoresis to detect the digestion status. If the digestion was complete, the gel recovery kit TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0 was used to recover the purified methyl parathion hydrolase gene fragment and the surface display plasmid fragment, and the recovery status was detected by 1.0% agarose gel electrophoresis.

Step 2: Connect the recovered methyl parathion hydrolase gene fragment and the surface display plasmid fragment. The connection system is as follows:

T ₄ DNA Ligase 1.0μL 10×T ₄ Buffer 2.0μL BtCSN gene fragment 8.0μL pINA1317-YICWP110 vector fragment 2.0μL Double distilled water 7.0μL 22℃ for 4h

Step 3: Transform E. coli DH5α with the ligation product, and finally take 100.0μl of the bacterial suspension and spread it on the LB screening plate containing kanamycin, culture at 37℃ for 16-18 h, pick the white spot clone and transfer it to 5.0 mL of LB liquid culture medium containing kanamycin, culture it at 37℃ overnight with shaking, use TIANprep Mini plasmid extraction kit to extract the plasmid, take 5.0μl for SfiI and BamHI double enzyme digestion verification, the enzyme digestion system is as follows:

50℃, overnight digestion;

Then, 0.5 μl of BamHI was added and the enzyme digestion was carried out at 37°C for 8 h. After the enzyme digestion was completed, 5.0 μl of the digestion product was taken and subjected to 1.0% agarose gel electrophoresis to detect the enzyme digestion status.

As a preferred technical solution of the present invention, the C-terminus of the fusion protein gene expressed by the plasmid of the surface display system of Yarrowia lipolytica has an anchor sequence at the CWP end and can be fixed on the surface of yeast cells.

As a preferred technical solution of the present invention, the recombinant plasmid pINA1317-YlCWP110-BtCSN is integrated into the Yarrowia lipolytica genome by non-homologous recombination through the zeta element carried on the plasmid for stable inheritance.

As a preferred technical solution of the present invention, the host bacteria Yarrowia lipolytica of the surface display plasmid pINA1317-YICWP110 is a food safety grade microorganism.

Compared with the prior art, the invention has the following beneficial effects: the strain can produce chitosanase BtCSN with higher activity, the surface display strain Yarrowia lipolytica of chitosanase BtCSN is a food safety-grade microorganism; the surface display system does not require induction when expressing exogenous genes, the used plasmid does not carry a resistance gene, and the plasmid can be recombined into the host genome for stable inheritance; the surface display technology is used to secrete and fix the chitosanase BtCSN on the cell surface, so as to solve the barrier problem of the cell membrane, enable the chitosan substrate and the enzyme to fully contact, avoid protein purification, and reduce costs; based on the surface display technology, the invention displays the chitosanase BtCSN on the cell surface of Yarrowia lipolytica, obtains a high-activity chitosanase transformant Y7, which can maintain 70% of its activity after being reused for 6 times, and has good application prospects; and preliminarily establishes the production process conditions for preparing chitosan oligosaccharides by whole-cell catalysis, laying a solid foundation for its large-scale preparation of chitosan oligosaccharides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart showing the construction of the surface display plasmid pINA1317-YICWP110-BtCSN of the present invention;

FIG2 is a diagram showing the results of PCR amplification of the BtCSN gene of the present invention;

FIG3 is a surface display plasmid pINA1317-YICWP110-BtCSN of the present invention;

FIG4 is a diagram showing the result of restriction enzyme digestion of the surface display plasmid pINA1317-YICWP110-BtCSNNotI of the present invention;

FIG5 is a graph showing the PCR verification results of the surface display strain of the present invention;

FIG6 is a curve diagram of enzyme activity determination according to the present invention;

FIG. 7 is a TLC analysis of the present invention showing the surface of the BtCSN transformant Y7 preparing chitosan oligosaccharide products;

1-10 represent the results of different enzymatic hydrolysis times (10min, 15min, 30min, 45min, 60min, 1.5h, 2h, 3h, 4h, 5h), and 11 represents the chitosan oligosaccharide standard with a polymerization degree of 1-7.

DETAILED DESCRIPTION

The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

Example 1: A tropical Bacillus, namely Bacillus tropicalis CSN-15, was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on May 23, 2022, with the deposit number: CGMCC No.24948, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named Bacillus tropicalis.

Obtaining the tropical Bacillus chitosanase gene BtCSN:

The specific steps are as follows:

(1) Activation of bacterial strains: Bacillus tropicus CSN-15 stored at -80°C was activated on a LB medium plate at 37°C for 18 h (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);

(2) Preparation of Bacillus tropicalis chitosanase BtCSN fragment: Bacillus tropicalusCSN-15 was used as a template and a BtCSN-F/R primer pair (BtCSN-F primer sequence: AGGCCGTTCTGGCCTACAACCTTCCAAACAACCTCAAG;

The primer sequence of BtCSN-R is: CGCGGATCCTGCCTTCAGACCTGCGACCAGCTT; the restriction sites at both ends are Sfi I and BamHI respectively) to amplify the full length of the BtCSN gene and the amplified product. The PCR system and procedure are as follows:

2×EsTaqMasterMix 25μL 10mMBtCSN-F 2.0μL 10mMBtCSN-R 2.0μL BacillustropicusCSN-15 DNA template 2.0μL Double distilled water 19μL total 50μL

Pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 45 s, for a total of 35 cycles; extension at 72°C for 5 min;

(3) The recovered gene fragment was connected to the pMD19-T simple vector using a T vector ligation kit (TaKaRapMD19-T simple Vector) to construct pMD19-T simple-BtCSN. The system and conditions are as follows:

Connect at 16°C for 16 h.

(4) Transform the recombinant plasmid into Escherichia coli DH5α;

1) Take 50.0 μl of DH5α competent cell suspension from the -80℃ refrigerator and place it on ice to thaw.

2) Add the DNA solution to be transformed (content not exceeding 50 ng, volume not exceeding 10 μl), gently rotate to mix, and place on ice for 30 minutes.

3) Place in a 42℃ water bath and heat shock for 90 seconds. Do not shake the tube during this period.

4) Quickly transfer the tube to ice and allow the cells to cool for 1-2 minutes.

5) Add 950 μl LB liquid culture medium, mix well and culture on a shaker at 37°C and 180 rpm for 45 min to allow the cells to recover and express the Amp resistance gene encoded by the plasmid.

6) Centrifuge the cultured bacterial solution at 5000×g for 5 minutes, remove 600 μl of the supernatant, resuspend the cells, and apply 100.0 μl of the cell suspension on a LA screening plate containing 40.0 μl of 20 mg/ml X-gal solution and 7.0 μl of 20% IPTG solution.

7) Place the plate upright in a 37°C incubator and culture for 30 minutes. After the bacterial solution is absorbed, invert the plate and continue to culture. Colonies can be observed after 12-16 hours.

8) Pick 10 single colonies and send them to Qingke Biotechnology Co., Ltd. for sequencing analysis and verification.

Example 2: Construction of surface display recombinant plasmid pINA1317-YICWP110-BtCSN;

The gene fragment of BtCSN was connected to the surface display plasmid to construct the recombinant plasmid pINA1317-YICWP110-BtCSN, as shown in Figure 1:

The specific experimental steps are as follows:

(1) Plasmids pMD19-T simple-BtCSN and pINA1317-YlCWP110 were double-digested with SfiI and BamHI, respectively. The digestion conditions and system were as follows:

50℃, overnight digestion.

Then add 2.0μl BamHI and digest at 37℃ for 8h. After the digestion is completed, take 5.0μl of the digestion product and check the digestion status by 1.0% agarose gel electrophoresis. If the digestion is complete, use a gel recovery kit (TaKaRa Agarose Gel DNA Purification Kit Ver.2.0) to recover the purified methyl parathion hydrolase gene fragment and surface display plasmid fragment, and check the recovery status by 1.0% agarose gel electrophoresis.

(2) Connect the recovered methyl parathion hydrolase gene fragment and the surface display plasmid fragment, and the connection system is as follows:

(3) Transform the ligation product into E. coli DH5α, and finally take 100.0μl of the bacterial suspension and spread it on the LB screening plate containing kanamycin, and culture it at 37℃ for 16-18h. Pick the white spot clone and transfer it to 5.0mL LB liquid culture medium containing kanamycin, and culture it at 37℃ overnight with shaking. Use TIANprep Mini plasmid extraction kit to extract the plasmid, and take 5.0μL for SfiI and BamHI double enzyme digestion verification. The enzyme digestion system is as follows:

50℃, overnight digestion.

Then, 0.5 μl of BamHI was added and digested at 37°C for 8 h. After the digestion was completed, 5.0 μl of the digestion product was taken and subjected to 1.0% agarose gel electrophoresis to detect the digestion status.

Example 3: Linearization of the recombinant surface display plasmid pINA1317-YICWP110-BtCSN;

Select the plasmid verified by double enzyme digestion in the previous step and the empty surface display plasmid pINA1317-YlCWP110 and digest them with restriction endonuclease NotI overnight in a 37°C water bath. The system is as follows:

NotI 2.0μL 10×GBuffer 10.0μL Plasmids 40.0μL Double distilled water 48.0μL

After the digestion is completed, 5.0 μl of the digestion product is taken and subjected to 1.0% agarose gel electrophoresis to detect the digestion status. If the digestion is complete, a gel recovery kit (TaKaRa Agarose Gel DNA Purification Kit Ver. 2.0) is used to recover and purify the large fragment containing the target gene and the empty surface display plasmid large fragment, and the recovery status is detected by 1.0% agarose gel electrophoresis.

Example 4: Construction of surface display recombinant strains;

The recovered and purified large fragment containing the target gene was used to transform Yarrowialipolytica Po1h, and the surface display plasmid fragment without BtCSN was transformed as a control. The yeast transformation was carried out using the lithium acetate method, and the steps were as follows:

(1) Streak out single colonies of the host bacteria Y. lipolytica Po1h on YPD solid medium and culture at 28°C for 2 days;

(2) Pick a single colony, inoculate it into 5.0 ml YPD liquid medium, and culture it at 28°C overnight;

(3) Inoculate the above bacterial solution into 50.0 ml YPD liquid medium at a final concentration of 5×10 ⁶ cells/ml and culture at 28°C until the cell concentration reaches 2×10 ⁷ cells/ml; (approximately 4 hours)

(4) Centrifuge the bacterial solution at 2500 × g for 5 min, discard the supernatant, and suspend the cells in 1.0 ml of TE buffer;

(5) Centrifuge the bacterial suspension at 3000×g for 5 min, discard the supernatant, and resuspend the cells in 600.0 μl of 0.1 M lithium acetate (pH 6.0) buffer.

(6) Place the cell suspension in a 28°C water bath for 1 h. After the water bath ends, centrifuge at 3000×g for 2 min and discard the supernatant.

(7) Gently resuspend the cells in 120.0 μl of 0.1 M lithium acetate (pH 6.0) buffer. At this point, the preparation of yeast Y.lipolytica Po1h competent cells is complete;

(8) Boil 1.0 ml of single-stranded fish sperm DNA (10 mg/ml) sample for 5 min and quickly cool on ice; (the purchased sample can be used directly, this step can be omitted)

(9) Take 40.0 μl of the above yeast competent cells, add 5.0 μl of single-stranded fish sperm DNA and 5.0 μl of the DNA fragment to be transformed, and incubate in a 28°C water bath for 15 min;

(10) Add 350.0 μl of 40% (w/v) PEG 4000 (dissolved in 0.1 M lithium acetate, pH = 6.0) and 16.0 μl of 1 M DDT to the above sample and incubate in a static water bath at 28°C for 1 h;

(11) Add 40.0 μl DMSO dropwise, mix gently, and heat shock at 39°C in a water bath for 10 min;

(12) Add 400.0 μl of 0.1 M lithium acetate (pH 6.0) buffer and mix gently;

(13) Spread the above bacterial suspension on YNB plates and culture at 28°C for 3-4 days.

Example 5: Transformant screening;

(1) PCR verification;

Randomly pick the transformants with yellow hydrolysis circles in the previous step, inoculate them into 5.0 ml YPD liquid culture and culture them at 28°C overnight, then use TIANGEN yeast genomic mini kit to extract the genomic DNA of the transformants. For specific operation methods, please refer to the instructions.

Then, the transformant genomic DNA was used as a template and the BtCSN-F/R primer pair was used for PCR verification. The PCR product was detected by agarose gel electrophoresis to confirm whether the BtCSN gene was recombined into the transformant genomic DNA.

(2) Determination of transformant enzyme activity (see Example 6 for the enzyme activity determination method);

The enzyme activity of the above-verified transformants was determined by the DNS method, and those with higher enzyme activity were used for subsequent experiments.

Example 6: Enzyme activity determination;

The enzyme activity determination method adopted is the DNS (3,5-dinitrosalicylic acid) method, with glucosamine as the standard. Specific steps: The experimental design is divided into three groups, namely the experimental group, the control group and the blank group. 0.1ml crude enzyme solution and 0.9ml colloidal chitosan solution were added to the EP tube of the experimental group, 0.1ml distilled water and 0.9ml colloidal chitosan solution were added to the EP tube of the blank group, and 0.9ml colloidal chitosan and 0.1ml bacterial solution after boiling water inactivation for 10 minutes were added to the control group. The three groups of EP tubes were placed in a 50℃ water bath at the same time for reaction for 15 minutes, then taken out and the reaction was terminated by boiling water bath for 10 minutes. Take out the three groups after inactivation, rinse with running water, cool and centrifuge. Then take 0.1 ml of each supernatant sample from the three groups after centrifugation and 0.2 ml of DNS solution into new EP tubes, boil them in a boiling water bath for 5 min, add 0.9 ml of distilled water, take 200 μL into a 96-well plate, measure the absorbance at 540 nm, perform parallel experiments on the three groups and take the average value to keep the standard error within 5%.

The amount of enzyme required to produce 1 μmol of chitosan oligosaccharide in 1 minute under the above conditions is one unit of enzyme activity. The enzyme activity unit (U) is defined as: the amount of enzyme (mL) required to catalyze the production of 1 μmol of reducing sugar per minute at 50°C under certain conditions.

Enzyme activity calculation formula: Enzyme activity (U/ml) = m×N×1000/(M×T×V);

m: amount of glucose (mg) obtained from the standard curve;

N: dilution multiple;

M: glucose molecular weight: 180.16;

T: reaction time;

V: enzyme solution volume;

Example 7: Optimization of whole-cell catalytic conditions of BtCSN surface display strains;

Use 6% (m/V) L chitosan solution, add 100U/mL enzyme, pH 6.0, 50℃ hydrolysis for 5h, boil in water for 15min to kill enzyme, add 5moL/L NaOH solution to adjust pH to greater than 9, centrifuge at 12000rpm/min for 15min with precipitated chitosan, freeze-dry the supernatant and precipitate, and weigh the mass. The hydrolysis rate and chitosan yield are calculated as follows:

Hydrolysis rate % = (1-m3/m1) × 100;

Chitosan oligosaccharide yield % = (m2-m4-m5) × 100/m1;

Wherein: m1 is the mass of chitosan; m2 is the mass of the supernatant after freeze-drying; m3 is the mass of the freeze-dried precipitate; m4 is the mass of acetic acid; m5 is the mass of NaOH.

(1) The effect of enzymatic hydrolysis time on the preparation of chitosan oligosaccharides by hydrolysis of chitosan;

The reaction system was reacted in a 50℃ constant temperature shaker for 1h, 2h, 3h, 4h, 5h, 6h, and 7h, respectively, and then the enzyme was inactivated in a boiling water bath for 15min. A 5moL/L NaOH solution was added to adjust the pH to greater than 9, and the precipitate was used as the reacted chitosan. The chitosan was centrifuged at 12000rpm/min for 15min, and the supernatant and precipitate were freeze-dried and weighed. The hydrolysis rate was calculated, and the effect of enzymatic hydrolysis time on the preparation of chitosan oligosaccharides by surface-displayed BtCSN transformant Y7 was analyzed.

(2) The effect of enzyme addition on the preparation of chitosan oligosaccharides;

The reaction system was added with enzymes of 30, 50, 70, 100, 120, and 150 U/ml cell suspension, respectively, and reacted in a constant temperature shaker at 50°C for 5 hours, then the enzyme was inactivated in a boiling water bath for 15 minutes, and 5 mol/L NaOH solution was added to adjust the pH to greater than 9. The chitosan was precipitated and centrifuged at 12000 rpm/min for 15 minutes. The supernatant and precipitate were freeze-dried and weighed. The hydrolysis rate and oligosaccharide yield were calculated, and the effect of enzyme addition on the preparation of chitosan oligosaccharides by surface display BtCSN transformant Y7 was analyzed.

(3) The effect of acetic acid amount on the preparation of chitosan oligosaccharides by hydrolysis of chitosan;

1%, 2%, 3%, 4%, and 5% acetic acid were added to the reaction system, respectively, and the reaction was carried out in a constant temperature shaker at 50°C for 5 hours, and then the enzyme was inactivated in a boiling water bath for 15 minutes, and a 5 mol/L NaOH solution was added to adjust the pH to greater than 9, and the chitosan was precipitated and centrifuged at 12000 rpm/min for 15 minutes. The supernatant and the precipitate were freeze-dried and weighed. The hydrolysis rate and oligosaccharide yield were calculated, and the effect of acetic acid on the preparation of chitosan oligosaccharides by hydrolyzing chitosan by surface display BtCSN transformant Y7 was analyzed.

(4) The effect of rotation speed on the preparation of chitosan oligosaccharides from chitosan;

The reaction system speed was set to 50, 100, 150, 180, 210, and 240 rpm/min, respectively. After reacting in a constant temperature shaker at 50°C for 5 hours, the enzyme was inactivated in a boiling water bath for 15 minutes, and a 5 mol/L NaOH solution was added to adjust the pH to greater than 9. The chitosan was precipitated and centrifuged at 12000 rpm/min for 15 minutes. The supernatant and precipitate were freeze-dried and weighed. The oligosaccharide yield was calculated, and the effect of the speed on the preparation of chitosan oligosaccharides by hydrolyzing chitosan by surface-displayed BtCSN transformant Y7 was analyzed.

(5) The effect of the number of times of use on the enzyme activity of surface displayed BtCSN;

Using the process optimized in the above steps (1-4), the surface-displayed BtCSN transformant Y7 was used six times to prepare chitosan oligosaccharides, and the enzyme activity was measured before and after the experiment.

(6) Analysis of prepared chitosan oligosaccharide products;

TLC method: The above enzymatic hydrolysis product is spotted on the marked chromatography plate with a pipette, and the layer is developed for about 1.5 hours using n-propanol: 25% ammonia water: water = 8:3:1. The chromatography plate is taken out, blown dry, and 0.1% ninhydrin solution (dissolved in ethanol) is prepared as a color developer and evenly sprinkled on the chromatography plate, placed in a 110°C oven for color development for about 10 minutes, and the product analysis is performed based on the color development results.

The experimental results are shown in FIG2 , where M is DL2000 DNA Ladder, and from top to bottom are 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp and 100 bp respectively; and 1 is the BtCSN gene.

As shown in Figure 2, PCR amplified a fragment of about 700 bp. The sequencing results showed that the full length of the BtCSN gene was 729 bp, encoding 242 amino acids. The specific sequence is as follows:

>BtCSN gene sequence 729bp

ATGGCTGGTCTGAACAAGGACCAGAAGCGACGAGCTGAACAGCTGACCT

CCATCTTCGAGAACGGAACTACCGAGATCCAGTACGGTTACGTTGAGCGA

CTTGACGATGGTCGAGGTTACACCTGTGGTAGAGCTGGTTTCACCACCGCT

ACTGGTGATGCTCTTGAGGTTGTCGAGGTCTACACCAAGGCTGTGCCTAAC

AACAAGCTGAAGAAGTACCTGCCTGAACTGCGAAGACTGGCTAAGGAGG

AGTCTGACGACACCTCTAACCTGAAGGGTTTCGCATCCGCATGGAAGTCTC

TTGCCAACGACAAAGAGTTCCGAGCAGCTCAAGACAAGGTCAACGACCA

CCTGTACTACCAGAACGCCATGAAGCGATCCGATAACGCTGGACTGAAGA

CCGCTCTTGCTCGAGCTGTCATGTACGACACCGTCATTCAGCATGGTGATG

GTGATGACCCTGACTCTTTCTACGCTCTGATCAAGCGAACCAACAAGAAG

GCTGGTGGTTCGCCTAAAGACGGCATTGACGAGAAGAAGTGGCTGAACA

AGTTTCTGGATGTCCGATACGACGACCTGATGAACCCTGCTAACCACGACA

CTCGAGACGAATGGCGAGAGTCTGTTGCTCGAGTTGACGTTCTGCGATCTA

TCGCCAAAGAGAACAACTACAACCTGAACGGACCTATTCACGTCCGATCCAACGAGTACGGTAACTTCGTCATCTAA;

Amino acid sequence 242aa:

MAGLNKDQKRRAEQLTSIFENGTTEIQYGYVERLDDGRGYTCGRAGFTTATG

DALEVVEVYTKAVPNNKLKKYLPELRRLAKEESDDTSNLKGFASAWKSLAN

DKEFRAAQDKVNDHLYYQNAMKRSDNAGLKTALARAVMYDTVIQHGDGD

DPDSFYALIKRTNKKAGGSPKDGIDEKKWLNKFLDVRYDDLMNPANHDTRDEWRESVARVDVLRSIAKENNYNLNGPIHVRSNEYGNFVI;

Figure 3 shows the surface of plasmid pINA1317-YICWP110-BtCSN: M is DL5000 DNA Ladder, from top to bottom are 5000bp, 4000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp; 1 is the result of enzyme digestion of pINA1317-YICWP110-BtCSN.

The above fragments were recombined into pINA1317-YICWP110 by restriction digestion and T4 DNA ligase, and the results of restriction digestion of the surface display plasmid pINA1317-YICWP110-BtCSN are shown in Figure 3. In band 1: the upper fragment of about 5000 bp is the pINA1317-YICWP110 fragment, and the lower fragment of about 700 bp is the BtCSN gene fragment.

The surface display plasmid pINA1317-YlCWP110-BtCSNNotI digestion results as shown in Figure 4

M is 1kb DNA ladder, from top to bottom are 10kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb; 1 is the result of pINA1317-YlCWP110-BtCSNNotI digestion;

The plasmid pINA1317-YlCWP110-BtCSN was linearized and digested with NotI. The digestion results are shown in Figure 4. The plasmid pINA1317-YlCWP110-BtCSN was digested into two fragments. The small fragment was about 2000 bp in size and was the kanamycin resistance region on the plasmid; the large fragment was a fragment of about 4500 bp in size containing the BtCSN gene fragment. This fragment was recovered and used for subsequent yeast transformation.

The PCR verification results of the surface display strains are shown in Figure 5; M is DL2000 DNA Ladder, and from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp respectively; 1-16 are positive transformants.

The transformant genomic DNA was used as a template to amplify the target band of about 700 bp, which was consistent with the actual size of the BtCSN gene fragment, indicating that the expression vector

The linear fragment of pINA1317-YlCWP110-BtCSN was successfully integrated into the genomic DNA of Y.lipolytica Po1h.

The chitosanase activity of the positive transformants was determined, and the glucose standard curve obtained by the DNS method was shown in Figure 6, with a linear equation of y = 0.1257x-0.0219 and R ² of 0.9992. Through this standard curve, the chitosanase activity of the surface-displayed strain Y7 was found to be the highest, at 317.53 ± 6.29 U/mg cells (bacterial solution 2836.66 ± 16.21 U/ml cells usage).

Effect of enzymatic hydrolysis time on the preparation of chitosan oligosaccharides by surface-displayed BtCSN transformant Y7;

The effect of enzymatic hydrolysis time on the hydrolysis rate is shown in Table 1. As the enzymatic hydrolysis time increases, the hydrolysis rate of chitosan gradually increases. When the enzymatic hydrolysis time continues to increase after reaching 5 h, the hydrolysis rate remains almost unchanged.

Table 1: Effect of enzymatic hydrolysis time on hydrolysis rate

Effect of enzyme dosage on the hydrolysis of chitosan to prepare chitooligosaccharides by surface-displayed whole cells transformant Y7;

The effect of the amount of enzyme added to the surface display whole cell Y7 strain on the oligosaccharide yield is shown in Table 2. With the increase of the amount of enzyme added, the oligosaccharide yield of chitosan gradually increased. When the enzyme was added at an amount of 120U/ml cell suspension, the oligosaccharide yield remained almost unchanged.

Table 2: Effect of enzyme addition amount on oligosaccharide yield

Effect of acetic acid amount on the preparation of chitosan oligosaccharides by hydrolyzing chitosan by surface-displayed BtCSN transformant Y7;

The effect of acetic acid on the yield of oligosaccharides is shown in Table 3. As the amount of acetic acid increases, the yield of oligosaccharides from chitosan gradually increases. When the amount of acetic acid is 2%, the acid concentration continues to increase, and the hydrolysis rate and oligosaccharide yield of 2% and 3% acetic acid remain almost unchanged. However, when the acetic acid content reaches 4% and 5%, the sugar yield and oligosaccharide yield drop sharply.

Table 3: Effect of acetic acid amount on oligosaccharide yield

Effect of rotation speed on the preparation of chitosan oligosaccharides by hydrolyzing chitosan by surface-displayed BtCSN transformant Y7;

The effect of rotation speed on oligosaccharide yield is shown in Table 4. As the rotation speed increases, the oligosaccharide yield of chitosan gradually increases. When the rotation speed is 180 rpm/min, the oligosaccharide yield does not change much.

Table 4: Effect of rotation speed on oligosaccharide yield

The process conditions for efficient preparation of chitosan oligosaccharides by surface display of BtCSN transformant Y7;

Considering the equipment, production cost and catalytic efficiency, the optimal enzymatic hydrolysis time was 4h, the enzyme dosage was 120U/mL, the acetic acid concentration was 2%, and the optimal speed was 180rpm/min. Under this condition, the hydrolysis rate was 95.3% and the oligosaccharide yield was 89.77%.

Effects of usage times on the enzyme activity of surface-displayed BtCSN transformant Y7

As shown in Table 5, after repeated use for 6 times, the enzyme activity of the surface-displayed BtCSN transformant Y7 was 1941.69 U/ml cellsuspension, with only a loss of 31.55%, which has good stability and application prospects.

Table 5: Effect of the number of uses on the enzyme activity of surface-displayed BtCSN transformant Y7

Product analysis by TLC:

As shown in Figure 7, the TLC analysis surface shows the analysis of chitosan oligosaccharide products prepared by BtCSN transformant Y7; 1-10 represent the results of different enzymatic hydrolysis times (10min, 15min, 30min, 45min, 60min, 1.5h, 2h, 3h, 4h, 5h), and 11 represents the chitosan oligosaccharide standard products with a polymerization degree of 1-7.

As the reaction proceeded, the thin layer chromatography analysis results of the enzymatic hydrolysis products of the surface-displayed BtCSN transformant Y7 were shown in Figure 7. No monomeric glucosamine was observed in the enzymatic hydrolysis products, indicating that it was an endo-chitosanase. As the enzymatic hydrolysis time increased after 4 hours, the oligosaccharide composition did not change much, and was mainly composed of chitosan oligosaccharides with a degree of polymerization of 3-7.

The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims (8)

1. A strain of bacillus tropicalis, characterized in that: as tropical bacillus CSN-15, it has been deposited in China general microbiological culture Collection center, with the accession number: CGMCC No.24948, the preservation address is North Star Xili No. 1 and No. 3 in the Chaoyang area of Beijing, and the classification is named as bacillus tropicalis Bacillus tropicus.

2. Use of the bacillus tropicalis of claim 1 for producing a higher activity of chitosan enzyme BtCSN, wherein: the specific production steps are as follows:

s1: activating strains: activating tropical bacillus Bacillus tropicus CSN-15 preserved at-80deg.C in LB culture medium plate at 37deg.C for 18 hr;

S2: preparation of bacillus tropicalis chitosanase BtCSN fragment: using bacillus tropicalis Bacillus tropicus CSN-15 as template, amplifying BtCSN gene full length by BtCSN-F/R primer pair, PCR system and procedure as follows:

pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 45s for a total of 35 cycles; extending at 72 ℃ for 5min;

S3: the recovered gene fragment was ligated to pMD19-T simple Vector using T Vector ligation kit TAKARA PMD-T simple Vector to construct pMD19-T simple-BtCSN in the following system and conditions:

Connecting at 16 ℃ for 16h;

S4: transforming the recombinant plasmid into escherichia coli DH5 alpha;

S41: taking 50.0 mu.l DH5 alpha competent cell suspension from a refrigerator at-80 ℃ and thawing the cell suspension on ice;

S42: adding a DNA solution to be converted, wherein the content of the DNA solution is not more than 50ng, the volume is not more than 10 μl, and the mixture is gently rotated and mixed uniformly and placed on ice for 30min;

s43: placing in a water bath at a temperature of 42 ℃ and heat-shocking for 90s, wherein the tube is not required to be rocked during the heat shock;

s44: rapidly transferring the tube to ice to cool the cells for 1-2min;

s45: adding 950 μl of LB liquid medium, mixing, shaking at 37deg.C with 180rpm for 45min, recovering cells, and expressing Amp resistance gene encoded by plasmid;

s46: centrifuging the cultured bacterial liquid at 5000 Xg for 5min, sucking 600. Mu.l of supernatant, re-suspending the cells, and coating 100.0. Mu.l of cell suspension on a LA screening plate with 40.0. Mu.l of 20mg/ml X-gal solution and 7.0. Mu.l of 20% IPTG solution;

S47: the plate is placed in a 37 ℃ incubator for culturing for 30min, after bacterial liquid is absorbed, the plate is inverted, and the culture is continued, and bacterial colonies can be observed after 12-16 h.

3. Use of bacillus tropicalis according to claim 2 for the production of a higher activity of the chitosan enzyme BtCSN, characterized in that: LB medium in step S1: 1% tryptone, 1% sodium chloride, 0.5% yeast extract, 2% agar powder, sterilizing at 120deg.C for 20min, and cooling to room temperature.

4. Use of bacillus tropicalis according to claim 2 for the production of a higher activity of the chitosan enzyme BtCSN, characterized in that: btCSN-F primer sequence in step S2:

AGGCCGTTCTGGCCTACAACCTTCCAAACAACCTCAAG；

BtCSN-R primer sequence: CGCGGATCCTGCCTTCAGACCTGCGACCAGCTT; the cleavage sites at the two ends are Sfi I and BamHI respectively.

5. The construction of the surface display method of the chitosan enzyme BtCSN according to claim 2, wherein: recombinant plasmids pINA1317-YICWP110-BtCSN are constructed by recombining chitosan enzyme BtCSN onto surface display plasmids pINA1317-YlCWP110 based on a surface display system of yarrowia lipolytica Yarrowia lipolytica;

The method comprises the following specific steps:

step 1: the plasmid pMD19-T simple-BtCSN and pINA1317-YlCWP110 were subjected to SfiI and BamHI double digestion respectively, and the digestion conditions and system were as follows:

Enzyme cutting at 50 ℃ overnight;

Then adding 2.0 mu lBamHI, after the enzyme digestion is finished, taking 5.0 mu l of enzyme digestion products, detecting enzyme digestion conditions by agarose gel electrophoresis of 1.0%, if the enzyme digestion is complete, respectively cutting gel to recover and purify methyl parathion hydrolase gene fragments and surface display plasmid fragments by using a gel recovery kit TaKaRa Agarose Gel DNA Purification KitVer.2.0, and detecting recovery conditions by agarose gel electrophoresis of 1.0%;

step 2: the recovered methyl parathion hydrolase gene fragment and the surface display plasmid fragment are connected, and the connection system is as follows:

Step 3: e.coli DH5 alpha is transformed by the connection product, finally 100.0 mu L of bacterial suspension is coated on an LB screening plate containing kanamycin, the culture is carried out for 16-18h at 37 ℃, white spot clones are picked up and transferred into 5.0mL of LB liquid culture medium containing kanamycin, the shake culture is carried out at 37 ℃ overnight, plasmids are extracted by using TIANPREP MINI plasmid small extraction kit, 5.0 mu L of the plasmid small extraction kit is taken for SfiI and BamHI step-by-step double enzyme digestion verification, and the enzyme digestion system is as follows:

Enzyme cutting at 50 ℃ overnight;

Then, 0.5. Mu. lBamHI was added thereto and the mixture was digested at 37℃for 8 hours, and after the digestion was completed, 5.0. Mu.l of the digested product was subjected to 1.0% agarose gel electrophoresis to detect the digestion.

6. The construction of the surface display method of chitosan enzyme BtCSN, wherein: the C-terminal of the plasmid-expressed fusion protein gene of yarrowia lipolytica Yarrowia lipolytica surface display system has a CWP terminal anchor sequence, which can be immobilized on the yeast cell surface.

7. The construction of the surface display method of chitosan enzyme BtCSN, wherein: the recombinant plasmid pINA1317-YlCWP110,110-BtCSN is integrated into the Yarrowia lipolytica genome by non-homologous recombination via the zeta element carried on the plasmid.

8. The construction of the surface display method of chitosan enzyme BtCSN, wherein: the host bacterium yarrowia lipolytica Yarrowia lipolytica with surface display of plasmid pINA1317-YlCWP110 is a food-safe microorganism.