CN108018245A - One plant of bacillus subtilis for producing chitosan enzyme and its application - Google Patents

Abstract

The invention belongs to the field of biotechnology, and in particular relates to a chitosanase-producing bacillus subtilis and an application thereof. The bacillus subtilis provided by the invention has a high yield of chitosanase, strong enzyme activity, and a different protein structure from common chitosanases, which provides a new idea for in-depth research on the molecular mechanism of chitosanases. Ordinary chitosanases only have good activity when they are weakly alkaline to weakly acidic, but the chitosanases found in the present invention have good activity under weakly alkaline, neutral, weakly acidic and strongly acidic conditions. active. Simultaneously, the chitosan enzyme that the present invention discovers can not only act on dissolving chitosan, but also can act on insoluble high polychitosan, omits the treatment before chitosan degradation; The final reaction product is two Polyglucosamine and tripolyglucosamine have simple ingredients, which are convenient for subsequent further processing and application, and greatly save production costs.

Description

A strain of Bacillus subtilis producing chitosanase and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a chitosanase-producing bacillus subtilis and an application thereof.

Background technique

Glucosamine is an important pharmaceutical precursor and chemical raw material, which has a wide range of uses. Chitosan derived from shrimp and crab shells is a polymer formed by connecting D-glucosamine (or a small amount of N-acetyl-D-glucosamine) through β-1,4-glycosidic bonds. Sugar exceeds 100 billion tons, and a large amount of glucosamine can be produced by hydrolyzing chitosan.

At present, the commonly used chitosan hydrolysis methods are: acid hydrolysis, oxidative degradation, physical degradation and enzymatic hydrolysis. Among them, enzymatic degradation of chitosan has the advantages of low environmental pollution, mild reaction conditions, and no by-products. It is currently the most widely used chitosan degradation method and has been gradually used in industrial production in recent years. Enzymatic hydrolysis of chitosan The key to sugar lies in chitosanase.

Although there are many advantages in the enzymatic preparation of chitosan oligosaccharides, the existing chitosanases are ubiquitous: the applicable pH range is narrow, they can only act on soluble chitosan, and the degradation products are mostly pentasaccharides (G5) to above oligosaccharides etc. For example, a report titled "Research Overview of Microbial Chitosanases" pointed out that most of the existing chitosanases are basic proteins, and the optimum pH is generally concentrated between 5 and 8, and occasionally a few The optimal pH of each enzyme is 4, but the corresponding optimal temperature is too high, resulting in high energy consumption and limited scope of application in actual production and application.

Therefore, the development of a chitosanase with a wide range of suitable pH and a relatively low optimum temperature, which can degrade insoluble chitosan into oligosaccharides such as disaccharides (G2) and trisaccharides (G3), can effectively promote The deep utilization of chitosan from marine shrimp and crab has important practical significance.

Contents of the invention

The purpose of the present invention is to provide a chitosanase-producing bacillus subtilis and application thereof.

In order to achieve the above-mentioned invention, the technical solution adopted in the present invention is: a strain of Bacillus subtilis, preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on October 30, 2017, strain name: Bacillus subtilis, Bacillus subtilis , MY002, the deposit number is CGMCC No.14841.

Correspondingly, for a strain of Bacillus subtilis, its 16SrDNA sequencing result is shown in SEQ ID NO 1; its gyrA sequencing result is shown in SEQ ID NO 2; its gyrB sequencing result is shown in SEQ ID NO 3.

Correspondingly, the application of the bacillus subtilis in hydrolyzing chitosan.

Preferably, the pH range of the application is 2.5-7.5.

Preferably, the pH of the application is: 3 or 6.

Preferably, the temperature range of the application is 35-65°C.

Correspondingly, a chitosanase CsnMY002 is characterized in that: its nucleotide sequence is shown in SEQ ID NO 4, and its amino acid sequence is shown in SEQ ID NO 5.

Correspondingly, the application of the chitosanase CsnMY002 in hydrolyzing chitosan.

Preferably, the pH range of the application is: 2.5-7.5.

Preferably, the application temperature is 35-65°C.

The present invention has the following beneficial effects:

1. The present invention provides a strain of Bacillus subtilis producing chitosanase. The chitosanase produced by it has the advantages of high yield and strong enzyme activity.

2. The chitosanase found in the present invention has a protein structure different from common chitosanases, and the amino acid sequence TRDEWR fragment of its protein is in a helical structure; while there is no such sequence in other chitosanases, the corresponding The location is an irregular Loop structure. This provides a new idea for the in-depth study of chitosanase molecular mechanism.

3. Ordinary chitosanases only have good activity when they are weakly acidic to weakly alkaline, but the chitosanases found in the present invention are not only active under conventional conditions, but also have good activity under stronger acidic conditions. enzyme activity. This makes the application range and prospect of the enzyme much larger than that of common chitosan enzymes.

4. The chitosanase found in the present invention can not only act on soluble chitosan, but also act on insoluble high polychitosan or even untreated crude chitosan raw material, omitting chitosan The treatment before degradation greatly saves the production cost.

5. The products of chitosan hydrolyzed by chitosanase found in the present invention are mainly diglucosamine and trimeric glucosamine, and the product components are simple, which is convenient for further processing and application.

Description of drawings

Fig. 1 is the hydrolysis circle blank control of the strain not inoculated;

Fig. 2 is the transparent hydrolysis circle that screening bacterial strain forms on selection medium;

Fig. 3 is D-glucose hydrochloride standard curve figure;

Fig. 4 is the 16SrDNA phylogenetic tree of strain MY002;

Figure 5 is the phylogenetic tree of gyrA of strain MY002;

Figure 6 is the phylogenetic tree of gyrB of strain MY002;

Fig. 7 is the chitosanase gel filtration chromatography result that removes signal peptide;

Fig. 8 is the effect comparison figure of chitosanase CsnMY002 at different temperatures;

Fig. 9 is a comparison chart of the effect of chitosanase CsnMY002 at different pHs;

Figure 10 is the analysis of chitosanase CsnMY002 hydrolyzed colloidal chitosan product;

Fig. 11 is the analysis of insoluble high-polychitosan products hydrolyzed by chitosanase CsnMY002.

Detailed ways

Embodiment 1: the screening of producing chitosanase bacterial strain

1. Material preparation:

(1) Soil samples: Soil samples were collected from the asparagus growing area in Mianyang.

(2) Medium:

1) Chitosanase screening medium (g/L): colloidal chitosan 2.0, K ₂ HPO ₄ 0.7, KH ₂ PO ₄ 0.3, (NH ₄ ) ₂ SO ₄ 5, MgSO ₄ . 7H ₂ O 0.5, FeSO ₄ . 7H ₂ 0 0.01, agar 12.0, pH=7.0;

2) Broth medium (g/L): peptone 10.0, beef extract 3.0, NaCl 5.0, pH=7.0.

3) Chitosanase induction medium (g/L): colloidal chitosan 5.0, K ₂ HPO ₄ 0.7, KH ₂ PO ₄ 0.3, (NH ₄ ) ₂ SO ₄ 1.0, MgSO ₄ . 7H ₂ O 0.5, FeSO ₄ . 7H ₂ 0 0.01, pH=7.0;

4) LB medium (g/L): peptone 10.0, yeast extract 5.0, NaCl 10.0.

(3) Reagents:

1) Colloidal chitosan: Weigh 10 g of high-polymer fine powder chitosan, add appropriate amount of concentrated hydrochloric acid, and stir on a magnetic stirrer for 4-6 hours to fully dissolve, then repeatedly wash and centrifuge with deionized water (4000r/min ), and finally form a jelly, while stirring, usually slowly add NaOH to adjust the pH to neutral to obtain colloidal chitosan, which is stored at 4°C for later use.

2) 3,5-dinitrosalicylic acid (DNS): Take 50ml of distilled water and heat it in a water bath at 80°C, add 26.2ml of 2mol/L NaOH and 0.63g of 3,5-dinitrosalicylic acid after heating base salicylic acid, after stirring and dissolving, add 18.2g potassium sodium tartrate, stir and dissolve, then add 0.5g crystalline phenol and 0.5g Na ₂ SO ₃ , stir and dissolve, after cooling, dilute to 100ml with distilled water, store in brown Store in a bottle at 4°C for later use.

(4) Kits, enzymes and cells

1) BamHI and SalI were purchased from New England Biolabs, Inc.;

2) The plasmid extraction kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.;

3) T4 ligase was purchased from Takara Bio.Inc.;

4) Competent cells DH5α and Escherichia coli BL21 are preserved in our laboratory.

2. Primary screening of strains

(1) Weigh 5g of soil sample into a conical flask (250m) filled with 45ml of distilled water, stir to dissolve, boil for 30min, then take the supernatant for gradient dilution (10 ^-1 ,10 ^-2 ,10 ^-3 , 10 ^-4 , 10 ^-5 in total 5 concentrations), 100ul each was spread in the chitosanase screening medium (two parallels were set for each gradient), and cultured in a constant temperature incubator at 37°C for 2-4d .

(2) Pick the colonies that grow on the selective culture plate and have obvious transparent hydrolysis circles around them, streak and inoculate them on a new selective culture plate, place them in a constant temperature incubator at 37°C for 2 days, and then pick the colonies to streak and inoculate. In this way, three generations of screening are carried out to ensure that pure strains are obtained.

(3) After 3 consecutive generations of purification, pick a single colony and culture it in beef extract peptone medium, then take 5ul and add it on a filter paper sheet with a diameter of 6mm. The hydrolysis circle is shown in Figures 1 and 2. Measure the size of the transparent circle, as shown in Table 1.

Table 1 The size of the hydrolysis circle produced by screening strains

(4) The strains that are pure and capable of producing transparent hydrolysis circles are preserved and stored at -80°C.

3. Re-screening of bacterial strains and determination of chitosanase activity

(1) draw 5umol/L D-glucose hydrochloride standard curve

Take 30 200ul centrifuge tubes and evenly divide them into 3 groups of equal amounts, as shown in Table 2, add 5 μmol/ml D-glucose hydrochloride standard solution, distilled water and 3,5-dinitrosalicylic acid respectively (DNS) reagent, prepared into D-glucose hydrochloride standard reaction solutions with different contents. Shake each centrifuge tube evenly, place it in a boiling water bath and heat it accurately for 5 minutes to develop color, and then cool it rapidly in an ice bath. After cooling fully, use a microplate reader to measure the absorbance at a wavelength of 540nm. Take the average value of the three sets of data and fill in Table 2. Use Excel to draw a standard curve, which is the D-glucose hydrochloride standard curve, as shown in Figure 3.

Table 2 D-glucose hydrochloride standard curve

(2) Enzyme activity measured by DNS method

1) Obtain bacterial strain fermentation liquid: culture the selected bacterial strain overnight with broth medium, as seed liquid, then inoculate the seed liquid with 5% inoculum in the chitosanase induction medium, at 37 ° C, 220 r / Cultivate for 2 days under the condition of 1 min, then centrifuge at 12000 g for 30 min, and take the supernatant.

2) Take 200ul of the supernatant in step 1) and mix it with 200ul of phosphate buffer containing 1.0% colloidal chitosan (6.1ml of 20mM Na ₂ HPO ₄ solution mixed with 3.9ml of 20mM NaH ₂ PO ₄ solution, pH=7.0) . Incubate at 37°C for 1 hour, then boil in a water bath for 10 minutes to terminate the reaction, then cool in an ice bath, and then add 400ul of DNS reagent. After boiling in water for 5 minutes, quickly cool in an ice bath, centrifuge at 12000 r/min for 5 minutes, take the supernatant and measure the absorbance (OD value) at a wavelength of 540 nm. Using the standard curve of D-glucose hydrochloride as a control, the release amount U of the reducing sugar was calculated: U=(3.6842*average absorbance value+0.3692)*2.

Definition of enzyme activity unit: Under the above conditions, the amount of enzyme required to produce 1umol reducing sugar per hour is defined as 1 enzyme activity unit.

The enzyme activity assay results of the screened strains are shown in Table 3.

Table 3 screens the enzyme activity size of strain

Embodiment 2: bacterial strain identification

1. 16SrDNA sequencing identification

Extract the genomic DNA of the strain to be tested, and then clone the 16SrDNA from the genomic DNA with the universal primers of bacterial 16SrDNA.

The general primers are:

27F: AGA GTT TGA TCC TGG CTC AG;

1492R:TAC GGT TAC CTT GTT ACG ACT T.

The PCR results were sent to Sangon Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared with BLAST on the Gene Bank website (http://www.nicbi.nlm.nih.gov/), and the gene sequences of the standard strains were found and downloaded. The sequencing results are shown as SEQ ID Shown in NO 1. The genetic distance between different strains was calculated using MEGA7.0, and the phylogenetic tree of bacteria was drawn, as shown in Figure 4.

Comparing the 16SrDNA of the tested strain with the Bacillus type strain, the genetic distances with Bacillus subtilis (Bacillus subtilis), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) are the closest, respectively: 0.002, 0.005 and 0.015.

2. Sequencing identification of gyrA and gyrB

The A subunit and B subunit of gyrase, as housekeeping proteins, are evolutionarily conserved, with about 0.7-0.8% gene evolution every 1 million years, which is faster than the 1% evolution rate of 16SrDNA every 50 million years , so it is often used to identify strains that are more closely related and cannot be distinguished by 16SrDNA. Also using the genomic DNA of the strain to be tested as a template to amplify the gyrase A and B genes of the bacteria.

(1) gyrA is amplified using universal primers, the primers are:

gyrA-F: 5'-CAG TCA GGA AAT GCG TAC GTC CTT-3';

gyrA-R: 5'-CAA GGT AAT GCT CCA GGC ATT GCT-3'.

The sequencing result of gyrA is shown in SEQ ID NO 2. The PCR sequencing results were compared with the gyrase A genes of the standard strains of different genera, and a phylogenetic tree was constructed using MEGA 7.0, and the genetic distance was calculated. The results are shown in Figure 5. Among them, the genetic distance between the strain to be tested and Bacillus subtilis was the closest, which was 0.010 (the genetic distances with other strains were all above 0.2).

(2) gyrB is a primer designed according to the conserved region of the gyrB sequence of several closely related species based on the identification results of 16SrDNA. The primers are:

gyrB-F: 5'-AAC AGC AAA GGC CTT CAC CA-3';

gyrB-R: 5'-GCA GAG TCA CCC TCT ACG ATA TA-3'.

The sequencing result of gyrB is shown in SEQ ID NO 3. The PCR sequencing results were compared with the gyrase B genes of the standard strains of different genera, and a phylogenetic tree was constructed using MEGA 7.0, and the genetic distance was calculated. The results are shown in Figure 6. The genetic distance between the tested strain and Bacillus subtilis (Bacillus subtilis) was the closest at 0.016, and the genetic distance with Bacillus subtilis subsp.spizizenii was 0.087 (the genetic distance with other strains was above 0.2).

3. Based on the above results, it was determined that the tested strain MY002 was Bacillus subtilis. The strain was deposited at the China General Microbiological Culture Collection Center on October 30, 2017. The strain name: Bacillus subtilis MY002, and the preservation number is CGMCC No. 14841.

Embodiment 3: construct recombinant chitosanase CsnMY002

1. Recombinant expression and purification of chitosanase

(1) Cloning and sequencing of chitosanase gene

Through the multiple sequence alignment of the known Bacillus subtilis chitosanase gene in Genebank (https://www.ncbi.nlm.nih.gov/genbank/), the primers were designed as follows:

F:5'-CGC GGA TCC ATG AAA ATC AGT ATG CAA AAA GC-3';

R: 5'-A CGC GTC GAC TTA TTT GAT TAC AAA ATT ACC G-3'.

The entire genome of the strain was extracted using a bacterial whole genome DNA extraction kit, and polymerase chain reaction (PCR) was carried out using this as a template, and the chitosanase gene (CsnMY002) of the strain was successfully cloned, and its gene nucleoside The acid sequence is shown in SEQ ID NO 4, and the protein amino acid sequence is shown in SEQ ID NO 5.

Sequencing results showed that the full-length chitosanase gene of Bacillus subtilis MY002 was 834bp, encoding a total of 277 amino acids. The above amino acid sequence was predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/), and the first 35 amino acids were found to be the signal peptide sequence.

The inventors have found that there is a special sequence in the amino acid sequence of CsnMY002: TRDEWR (corresponding to the sequence in SEQ ID NO 5: Thr Arg Asp Glu Trp Arg), which forms a helical structure; but there is no such sequence in other chitosan enzymes Segment sequence, its corresponding position is an irregular Loop structure.

(2) Construction of recombinant plasmids

Using PET-28a as a vector, a recombinant plasmid (with 6 histidine tags at the N-terminal of the protein) was constructed without the signal peptide chitosanase. Primers were designed as follows:

F: 5'-CGC GGA TCC GCG GGA CTG AAT AAA GAT CAA AAG-3';

R: 5'-A CGC GTC GAC TTA TTT GAT TAC AAA ATT ACC G-3'.

Wherein, the underlined bases in the above primers were respectively introduced into the restriction sites of BamHI (GGA TCC) and SalI (GTC GAC). And the whole genome DNA of Bacillus subtilis MY002 was used as a template for PCR reaction. PCR products were recovered using a PCR product recovery kit.

At the same time, use the plasmid extraction kit to extract the PET-28a plasmid, put it and the PCR recovered product at 37°C overnight, carry out the BamHI and SalI double enzyme digestion reaction, and then digest the recovered product according to gene:plasmid=10: 1 (molar ratio) to construct a ligation system, and use T4 ligase to ligate at 4°C for 16 hours. Then the ligation system was transferred into the competent cell DH5α, and after the positive clones were selected, the plasmid was transferred into the expression host Escherichia coli BL21.

(3) Expression and purification of recombinant chitosanase (CsnMY002)

Inoculate the above-mentioned Escherichia coli BL21 strain containing the plasmid in LB medium containing 60ug/ml kanamycin, cultivate it at 37°C and 220rpm until the OD600 is 0.4-0.6, then cool down to 16°C, and add a final concentration of 0.2mM IPTG was carried out at low temperature overnight to induce the expression of recombinant chitosanase. Then 5600r/min, centrifuge for 30min to collect the bacteria.

Prepare a lysis buffer (50mM MES, 500Mm NaCl, 20mM imidazole, pH=6.0), suspend the precipitate with it, and then use ultrasound to disrupt the cells. After the disruption, the solution is centrifuged at 12000rpm at 4°C for 30min, and the supernatant is collected for Ni Column affinity chromatography, followed by elution with elution buffer (50 mM MES, 500 Mm NaCl, 100 mM imidazole, pH=6.0). The eluate obtained by affinity chromatography was then subjected to ion exchange chromatography using a Resource S cation column, and gel filtration chromatography using a Superdex 75 gel filtration column. Finally, a stable and active high-purity chitosanase was obtained.

The chitosanase gel filtration chromatography results without the signal peptide are shown in Figure 7, the relative molecular weight of the enzyme is 27.42kDa.

Cultured overnight in shake flasks, then purified by nickel column affinity chromatography. The yield of the enzyme is up to 100mg/L. At 37° C. and pH=6.0, the enzyme activity was 547±23 U/mg (as shown in Table 4).

The enzyme activity determination result of table 4 recombinant chitosanase

It can be seen from the above results that the recombinant chitosanase CsnMY002 has high yield and high activity.

Embodiment 4: the optimum reaction condition analysis of Bacillus subtilis MY002 chitosanase

1. Optimal reaction temperature analysis

(1) Preparation buffer: 50mM MES buffer (pH=6)

(2) Enzyme preparation: the concentration of the purified recombinant chitosanase CsnMY002 described in Example 3 was determined by Bradford method. 31.25 μg of enzyme solution was taken and made up to 500 μl with buffer solution (50 mM MES, pH=6), and the final concentration of enzyme preparation was 0.0625 μg/μl.

(3) Preparation of substrate solution: take 10ml of colloidal chitosan (1%, w/v), centrifuge (12000g, 5min) and remove supernatant, add 9.5ml of buffer solution (50mM MES, pH=6).

(4) Determination: Take 24 centrifuge tubes, each tube has a reaction system of 400 μl, and add 380 μl of substrate solution and 20 μl of enzyme preparation to each tube respectively. Set 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, a total of 8 temperature treatments, and set 3 parallels for each treatment. Each treatment was incubated at each temperature gradient for 5 min, and then boiled in boiling water for 10 min to terminate the reaction. After cooling down, add 400 μl DNS respectively, cook in boiling water for 5 minutes to develop color, and cool down rapidly. After cooling, centrifuge at 12000 g for 5 min to remove the chitosan precipitate.

(5) Result: each reaction system gets 200 μ l supernatant to measure absorbance value, then calculates the release amount of reducing sugar according to the D-glucose hydrochloride standard curve, thus reflects the recombinant chitosanase CsnMY002 at each temperature Hydrolytic activity. The result is shown in Figure 8.

The results showed that the recombinant chitosanase CsnMY002 had good enzyme activity at 35℃～65℃, and the optimum reaction temperature was 50℃. For the convenience of operation, the temperature in the following tests is all 37°C; in actual production, the required temperature can be selected according to the situation.

2. Analysis of optimum pH

(1) Prepare buffer: 50mM glycine-hydrochloric acid buffer (pH=2-4), 50mM MES buffer (pH=5-6), 50mM Tris-HCl buffer (pH=7-9).

(2) According to the method in step 1, the MES buffer solution was replaced by the above three groups of buffer solutions, and three groups of enzyme preparations and substrate solutions with different pH values were prepared, corresponding to three pH treatments, and each treatment was set up in three parallel . And according to the determination method of step 1, react at 37°C, calculate the release amount of reducing sugar, and obtain the hydrolysis activity of recombinant chitosanase CsnMY002 at different pH. The result is shown in Figure 9. It can be seen from the figure that the recombinant chitosanase CsnMY002 has good activity at pH=2.5-7.5, and the activity is the best at pH=3; the activity is excellent at pH=6. This is obviously different from the reported high activity range of chitosanase mainly concentrated at pH=5-7. The recombinant chitosanase CsnMY002 of the invention can not only hydrolyze chitosan substrates under weakly alkaline, neutral and weakly acidic conditions, but also act under stronger acidic conditions.

Embodiment 5: Analysis of hydrolysis performance of recombinant chitosanase CsnMY002

1. With soluble chitosan as substrate.

(1) Add 90 μl of colloidal chitosan solution (1%, w/v) to 7 centrifuge tubes, centrifuge at 12000 g for 5 min, remove supernatant, and resuspend with 50 mM, pH=6 MES buffer. Then add the chitosanase CsnMY002 solution (50mM, pH=6 MES damping fluid preparation) that concentration is 0.5mg/ml in 6 centrifuge tubes wherein, as experimental group; Add equivalent 50mM, pH=6 in 1 centrifuge tube MES buffer as a blank group.

(2) The above-mentioned experimental groups were subjected to hydrolysis reactions at 37° C. for 30 s, 1 min, 3 min, 5 min, 10 min, and 1 h; the blank group was placed under the same conditions for 1 h. Then boil in boiling water for 10min to terminate the reaction.

(3) Spot the G1-G6 mixed standard product on a silica gel plate (Silica gel 60 F ₂₅₄ , 1.05554.0001, purchased from Merck, Germany) as a standard reference (std in Figure 10); after the reaction is completed, Then, 1 μl of the centrifuged supernatant was taken from each treatment, and spotted on the above-mentioned silica gel plate for TLC analysis. The development system used by TLC is: ethyl acetate/ethanol/water/28% ammonia water=5/5/4/0.5 (v/v), after about 2 hours, it is completely developed, and the silica gel plate is air-dried and then immersed in 0.1% nendin Ketone (the solvent is ethanol), and then baked at 90° C. for 5 minutes to develop color. The hydrolysis results are shown in Figure 10.

(4) The results show that G3, G4, G5, and G6 can be detected after about 30 seconds of reaction. As the reaction progresses, longer chitosan oligosaccharides are gradually hydrolyzed into shorter oligosaccharides, and the final hydrolyzed products are G2 and G3. .

Wherein, G1, G2, G3, etc. mentioned in the whole text all represent oligosaccharides (oligosaccharides), and the numbers after G represent the degree of polymerization of glucosamine. Such as: G1 is glucosamine, G2 is dipolyglucosamine, G3 is tripolyglucosamine.

2. Use insoluble high polychitosan as substrate.

The operation steps are the same as step 1. Wherein, the equivalent amount of colloidal chitosan solution is replaced by insoluble chitosan suspension, and its preparation method is: take powdered chitosan 0.2g, add 10ml buffer (50mM MES, pH=6.0); fully shake, Suspend the powder. No blank group was set up, and the hydrolysis time was 4h, 8h, and 12h, respectively. The hydrolysis results are shown in Figure 11.

The results showed that the hydrolysis of insoluble high polychitosan by recombinant chitosanase was relatively slow, and the formation of hydrolysis products was detected after overnight incubation, and the products were still G2 and G3.

The above results show that the chitosanase CsnMY002 discovered in the present invention can not only hydrolyze soluble chitosan, but also hydrolyze insoluble high polychitosan. At the same time, unlike the final products of conventional chitosanases, which are generally G5, G6 or even longer-chain oligosaccharide mixtures, the final products of hydrolysis of chitosanase CsnMY002 are all short-chain sugars of G3 and G2, and the composition is more It is simple and convenient for subsequent applications, which may be the result of the special protein structure of the enzyme itself.

The chitosanase discovered in the present invention has a novel protein structure, has lower requirements on the working environment, and has an action effect different from other chitosanases, has a broader application prospect, and is also the molecular mechanism of chitosanase. A more in-depth study provides the theoretical basis.

sequence listing

<110> Chengdu Institute of Biology, Chinese Academy of Sciences

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atcttgggac gcagcggtat ccggaaagca tacgaatcag gccgaggctc tatcacgatc 660

cgggcaaaag ctgagatcga acaaacatct tcgggtaaag aaagaattat cgttacagag 720

ttaccttacc aagtgaataa ggcgaaatta attgagaaaa ttgctgatct cgtaagggac 780

aaaaagatag agggtatcac agatctgcgt gatgagtcag atcgtacagg tatgagaatt 840

gtcattgaaa tcagacgcga cgccaatgca aatgtcatct taaacaatct gtacaaacaa 900

actgctctac aaacatcttt tggcatcaac ctgcttgcac ttgtgatggc cagccgaaa 959

<210> 3

<211> 1142

<212>DNA

<213> Bacillus subtilis MY002

<400> 3

ccggagcatg tcgaatagta ttgacgaagc ccttgccggt tattgtacgg atatcaatat 60

ccaaatcgaa aaagacaaca gtatcacggt tgtagataat ggccgcggta ttccagtcgg 120

tattcatgaa aaaatgggcc gtcctgcggt agaagtcatt atgacggtgc ttcatgccgg 180

aggaaaattt gacggaagcg gctataaagt atccggagga ttacacggtg taggtgcttc 240

ggtcgtaaac gcactatcca cagagcttga tgtgacggtt caccgtgacg gtaaaattca 300

ccgccaaacc tataaacgcg gagttccggt tacagacctt gaaatcattg gcgaaacgga 360

tcatacagga acgacgacac attttgtccc ggaccctgaa attttctcag aaacaaccga 420

gtatgattac gatctgcttg ccaaccgcgt gcgtgaatta gcctttttaa caaagggcgt 480

aaacatcacg attgaagata aacgtgaagg acaagagcgc aaaaatgaat accattacga 540

aggcggaatt aaaagttatg tagagtattt aaaccgctct aaagaggttg tccatgaaga 600

gccgattac attgaaggcg aaaaggacgg cattacggtt gaagtggctt tgcaatacaa 660

tgacagctat acaagcaaca tttactcgtt tacaaacaac attaacacgt acgaaggcgg 720

tacccatgaa gctggcttca aaacgggcct gactcgtgtt atcaacgatt acgccagaaa 780

aaaagggctt attaaagaaa atgatccaaa cctaagcgga gatgacgtaa gggaagggct 840

gacagcgatt atttcaatca aacaccctga tccgcagttt gagggccaaa caaaaacaaa 900

gctgggcaac tcagaagcac ggacgatcac cgatacgtta ttttctacag cgatggaaac 960

atttatgctg gaaaatccag atgcagccaa aaaaattgtc gataaaggct taatggcggc 1020

aagagcaaga atggctgcga aaaaagcccg tgaactaaca cgccgtaaga gtgctttgga 1080

aatttccaac ttgcccggta agttagcgga ctgctctcaa aagatccgag catccccgag 1140

tg 1142

<210> 4

<211> 834

<212>DNA

<213> Bacillus subtilis chitosanase (CsnMY002)

<400> 4

atgaaaatca gtatgcaaaa agcagatttt tggaaaagg cagcgatctc attacktgtt 60

ttcaccatgt tttttaccct gataatgagc gaaacggttt ttgcggcggg actgaataaa 120

gatcaaaagc gccgggcgga acagctgaca agtatctttg aaaacggcac aacggagatc 180

caatatggat atgtagagcg attggatgat gggcgaggct atacatgcgg acgggcaggc 240

tttacaacgg ctaccgggga tgcattggaa gtagtggaag tatacacaaa ggcagttccg 300

aataacaaac tgaaaaagta tctgcctgaa ttgcgccgtc tggccaagga agaaagcgat 360

gatacaagca atctcaaggg attcgcttct gcctggaagt cgcttgcaaa tgataaggaa 420

tttcgcgccg ctcaagacaa agtaaatgac catttgtatt atcagcctgc catgaaacga 480

tcggataatg ccggactaaa aacagcattg gctagagctg tgatgtacga tacggttatt 540

cagcatggcg atggtgatga ccctgactct ttttatgcct tgattaaacg tacgaacaaa 600

aaagcgggcg gatcacctaa agacggaata gacgagaaga agtggttgaa taaattcttg 660

gacgtacgct atgacgatct gatgaatccg gccaatcatg acacccgtga cgaatggaga 720

gaatcagttg cccgtgtgga cgtgcttcgc tctatcgcca aggagaacaa ctataatcta 780

aacggaccga ttcatgttcg ttcaaacgag tacggtaatt ttgtaatcaa ataa 834

<210> 5

<211> 277

<212> PRT

<213> Bacillus subtilis chitosanase (CsnMY002)

<400> 5

Met Lys Ile Ser Met Gln Lys Ala Asp Phe Trp Lys Lys Ala Ala Ile

1 5 10 15

Ser Leu Leu Val Phe Thr Met Phe Phe Thr Leu Ile Met Ser Glu Thr

20 25 30

Val Phe Ala Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln

35 40 45

Leu Thr Ser Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr

50 55 60

Val Glu Arg Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly

65 70 75 80

Phe Thr Thr Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr

85 90 95

Lys Ala Val Pro Asn Asn Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg

100 105 110

Arg Leu Ala Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe

115 120 125

Ala Ser Ala Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala

130 135 140

Gln Asp Lys Val Asn Asp His Leu Tyr Tyr Gln Pro Ala Met Lys Arg

145 150 155 160

Ser Asp Asn Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr

165 170 175

Asp Thr Val Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr

180 185 190

Ala Leu Ile Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp

195 200 205

Gly Ile Asp Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr

210 215 220

Asp Asp Leu Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg

225 230 235 240

Glu Ser Val Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn

245 250 255

Asn Tyr Asn Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly

260 265 270

Asn Phe Val Ile Lys

275

Claims (10)

1. A strain of Bacillus subtilis, characterized in that it was preserved on October 30, 2017 in the General Microbiology Center of China Committee for the Collection of Microorganisms, strain name: Bacillus subtilis, Bacillus subtilis, MY002, and the preservation number is CGMCC No. 14841. 2. A strain of Bacillus subtilis, characterized in that: its 16SrDNA sequencing result is as shown in SEQ ID NO 1; its gyrA sequencing result is as shown in SEQ ID NO 2; its gyrB sequencing result is as shown in SEQ ID NO 3 . 3. the application of the bacillus subtilis described in claim 1 or 2 in hydrolyzing chitosan. 4. The application of Bacillus subtilis in hydrolyzing chitosan as claimed in claim 3, characterized in that: the pH range of the application is: 2.5-7.5. 5. the application of Bacillus subtilis in hydrolyzing chitosan as claimed in claim 3, is characterized in that: the pH=3 or 6 of described application. 6. The application of Bacillus subtilis in hydrolyzing chitosan as claimed in claim 3, characterized in that: the temperature range of the application is: 35-65°C. 7. A chitosanase CsnMY002, characterized in that its nucleotide sequence is shown in SEQ ID NO 4, and its amino acid sequence is shown in SEQ ID NO 5. 8. the application of chitosanase CsnMY002 described in claim 7 in hydrolyzing chitosan. 9. The application of chitosanase CsnMY002 in hydrolyzing chitosan as claimed in claim 8, characterized in that: the pH range of the application is: 2.5-7.5. 10. The application of chitosanase CsnMY002 in hydrolyzing chitosan according to claim 8, characterized in that: the temperature range of the application is: 35-65°C.