A recombinant chitosanase strain and its application in the preparation of chitosan oligosaccharides

Abstract

The present invention discloses a chitosanase gene derived from Glomerella Csn1 , expression vector, recombinant strain, method and application of using the chitosanase produced by the recombinant strain in degrading chitosan to prepare chitosan oligosaccharides. The present invention obtains a recombinant Pichia pastoris engineered strain capable of efficiently expressing the gene by recombining the chitosanase gene in the genus Glomeromyces into the Pichia pastoris engineered bacteria. The recombinant Pichia pastoris engineered strain that produces chitosanase has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO.M 20221107 and a deposit date of July 14, 2022. The chitosanase produced by the recombinant Pichia pastoris engineered strain of the present invention has high purity and low purification cost. The product of hydrolyzing chitosan is chitotriose, which has great application value. It is expected that the chitosanase of the present invention can be prepared in large quantities for the mass production of chitotriose, and has practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a chitosanase recombinant strain and application thereof in the preparation of chitosan oligosaccharides. Background Art

[0002] With the recent expansion of the global seafood market and the development of the aquatic product processing industry, the management of the millions of tons of shellfish waste generated annually has become a major issue. The use of shellfish waste to produce high-value chitosan and chitosan oligosaccharides has garnered significant attention in academia. Chitosan can be produced by deacetylation of chitin.

[0003] Chitooligosaccharides (COS) are oligosaccharides with a degree of polymerization of less than 20 produced by the hydrolysis of chitosan. They have a small molecular weight, can be absorbed by the gastrointestinal tract, and have higher solubility and lower viscosity. Their physiological activity is better than that of chitosan, and they have functional activities such as antibacterial and anti-inflammatory, anti-cancer and anti-tumor, antioxidant activity, and hypoglycemic activity. They are widely used in biomedicine, bioengineering, regenerative medicine, food industry, chemical industry, cosmetics, health products, and agriculture.

[0004] Studies have shown that the efficacy of chitosan oligosaccharides is closely related to their average molecular weight, degree of polymerization, and degree of acetylation, so it is very important to prepare chitosan oligosaccharide monomers with a specific degree of polymerization. For example, chitotriose is used to treat inflammatory bowel disease (Hu K, Yuan X, HeH, et al. Pharmacological mechanisms of chitotriose as a redox regulator inthe treatment of rat inflammatory bowel disease[J]. Biomedicine&Pharmacotherapy, 2022, 150: 112988.), promote peripheral nerve regeneration (Jiang M, Guo Z, Wang C,et al. Neural activity analysis of pure chito-oligomer components separatedfrom a mixture of chitooligosaccharides[J]. Neuroscience Letters, 2014, 581:32-36.), improve Parkinson's disease (Wang B, Wang L, Qu Y, et al. Chitosan oligosaccharidesexert neuroprotective effects via modulating the PI3K / Akt / Bcl-2 pathway in aParkinsonian model[J]. Food&Function, 2022, 13(10): 5838-5853.), and lower lipids (Shen X, Liang X, JiPreparation and antibacterial effect of chitooligosaccharidesmonomers with different polymerization degrees from crab shell chitosan byenzymatic hydrolysis[J]. Biotechnology and Applied Biochemistry, 2022. doi:10.1002 / bab.2339.), and antioxidant (Chen AS, Taguchi T, Sakai K, et al. Antioxidant activities of chitobiose and chitotriose[J]. Biological and Pharmaceutical Bulletin, 2003, 26(9): 1326-1330.) have great application prospects.

[0005] Chitosan oligosaccharide preparation methods are mainly divided into physical, chemical, and enzymatic degradation methods. Physical methods require harsh conditions, have low production efficiency, and the degree of polymerization of the produced chitosan oligosaccharide molecules is uncontrollable. Chemical methods are also unable to produce chitosan oligosaccharides with a uniform degree of polymerization, and harmful substances are introduced during the hydrolysis process, which greatly limits the application of chitosan oligosaccharide products in the biopharmaceutical field. Enzymatically produced chitosan oligosaccharides have better biological activity and, compared to traditional chemical and physical methods, have advantages such as mild conditions, controllable hydrolysis process, high product activity, and environmental friendliness.

[0006] Chitosan can be hydrolyzed by non-specific enzymes such as lipase, protease, chitinase, lysozyme and cellulase, but these enzymes have problems such as high enzyme consumption, poor specificity, low hydrolysis efficiency, incomplete degradation and difficult product separation during the degradation process.

[0007] Chitosanase (EC 3.2.1.132) is an enzyme that specifically hydrolyzes chitosan and can be used to hydrolyze chitosan to obtain high-purity chitosan oligosaccharides. However, most chitosanases produce chitosan oligosaccharides with varying degrees of polymerization, adding additional separation steps to obtain bioactive chitosan oligosaccharide monomers. Therefore, finding chitosanases that can cost-effectively and efficiently produce chitosan oligosaccharides with specific degrees of polymerization or acetylation will greatly promote the development of the chitosan oligosaccharide industry. Summary of the Invention

[0008] The technical problem to be solved by the present invention is how to solve the problems that the chitosan oligosaccharides produced by the existing chitosanase hydrolysis of chitosan have a non-uniform degree of polymerization, multiple separation steps and low production efficiency.

[0009] The present invention solves the above technical problems through the following technical means:

[0010] In a first aspect of the present invention, a chitosanase is provided, whose amino acid sequence is shown in SEQ ID No: 1, and the nucleotide sequence of the gene encoding the chitosanase is shown in SEQ ID No: 2.

[0011] It should be understood that those skilled in the art can, based on the amino acid sequence disclosed in the present invention, replace, delete and / or add one or more amino acids without seriously affecting its activity to obtain a mutant sequence of the protein. Therefore, the chitosanase of the present invention also includes proteins derived from the protein of SEQ ID No: 1 by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No: 1 and having equivalent activity. For example, proteins derived by adding a tag sequence, such as a His-tag or GST-tag, to the end.

[0012] Preferably, the amino acid sequence of the chitosanase-derived protein has a similarity of more than 70% to the amino acid sequence shown in SEQ ID No: 1, preferably more than 80%, and more preferably more than 90%.

[0013] Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode identical proteins. In addition, it will be appreciated that those skilled in the art can use conventional techniques to carry out nucleotide substitutions that will not affect the protein sequence encoded by the polynucleotides used in the present invention. In addition, methods known in the art can also be used to modify polynucleotides to enhance the activity or survival of the polynucleotides of the present invention in vivo.

[0014] The second aspect of the present invention provides a recombinant expression vector of the chitosanase gene.

[0015] Preferably, the recombinant expression vector is pPIC9K- Csn1 Expression vector.

[0016] The third aspect of the present invention provides a recombinant expression strain containing the above-mentioned recombinant expression vector.

[0017] Preferably, the recombinant expression strain is a recombinant Pichia pastoris engineered strain obtained by transferring the above recombinant expression vector into Pichia pastoris. Pichia pastoris GS115 / pPIC9K- Csn1The recombinant Pichia pastoris engineered bacteria has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO. M 20221107 and the deposit date July 14, 2022.

[0018] A fourth aspect of the present invention provides a method for obtaining the above-mentioned recombinant strain, comprising the following steps:

[0019] (1) Cloning of chitosanase gene: RNA of Trichoderma sphaeroides was extracted and cDNA synthesized by reverse transcription of RNA was used as template to amplify the chitosanase gene of the present invention. Csn1 ;

[0020] (2) Construction of cloning vector: The chitosanase gene obtained in step (1) Csn1 Introduced into T1-Simple vector and transformed into E. coli Trans1 T1 competent cells were screened and verified to obtain Trans1 T1 / T1 Simple- Csn1 Positively transformed strains;

[0021] (3) Construction of expression vector: The Trans1 T1 / T1 Simple- Csn1 The plasmid extracted from the positive transformation strain was used as a template, and the chitosanase gene was ligated into the chitosanase gene by double enzyme digestion. Csn1 Inserted into the downstream of pPIC9K promoter AOX1, pPIC9K- Csn1 The expression vector was transformed into Trans1 T1 competent cells, and the Trans1 T1 / pPIC9K- Csn1 Positively transformed strains;

[0022] (4) Obtaining the Pichia pastoris expression strain of chitosanase: The Trans1 T1 / pPIC9K- Csn1 The plasmid of the positive transformation strain is linearized and the linearized recombinant expression vector is transferred into the host by electroporation. Pichia pastoris In GS115, a recombinant Pichia pastoris strain producing chitosanase was screened and obtained. Pichia pastoris GS115 / pPIC9K- Csn1 .

[0023] Preferably, the double enzyme digestion and ligation method in step (3) includes: using Bln I (i.e. Avr II) and Not I two restriction enzymes to the chitosanase gene Csn1 Then, pPIC9K was ligated with T4 DNA ligase. Csn1connect.

[0024] In a fifth aspect, the present invention provides a method for preparing chitosan oligosaccharides by degrading chitosan using chitosanase expressed by the above-mentioned recombinant Pichia pastoris engineered strain, comprising the following steps:

[0025] (1) Expression of chitosanase: The recombinant Pichia pastoris engineered strain was inoculated into a 5 mL test tube containing BMGY medium for activation culture, and then transferred to BMGY medium for culture. The yeast cells were collected and cultured in BMM medium to induce chitosanase expression, and chitosanase was collected;

[0026] (2) Preparation of chitosan: chitosan is produced by enzymatically hydrolyzing the colloidal chitosan with the chitosanase obtained in step (1).

[0027] Preferably, the recombinant Pichia pastoris engineered strain is o The strain was induced to produce chitosanase by inducing the culture medium at 200 rpm and 0.5% (v / v) methanol was added to the culture medium every 24 h. After 7 d of induction culture, the chitosanase activity against 1% (w / v) colloidal chitosan reached 28.48 U / mL, and the specific activity reached 59.5 U / mg.

[0028] Preferably, in step (2), the enzymatic hydrolysis conditions are: substrate colloid chitosan concentration 0.25%~1.5% (w / v), pH 3.5~6.0, temperature 30~70 o C, reaction time is more than 0.5 h.

[0029] The advantages of the present invention are:

[0030] 1. The present invention obtains the coding of Coccidioides by PCR Gongronella sp. w5 chitosanase gene, and recombined it into Pichia pastoris to obtain a recombinant Pichia pastoris strain that can efficiently express the enzyme Pichia pastoris GS115 / pPIC9K- Csn1 , which has broadened resources for the discovery and application of new enzymes.

[0031] 2. The amino acid sequence of the chitosanase discovered in the present invention is shown in SEQ ID No: 1, which has the highest identity of 44.48% with the reported chitosanase amino acid sequence.

[0032] 3. After the recombinant Pichia pastoris engineered strain of the present invention is induced by methanol in BMM culture medium, the chitosanase expressed is of high purity and does not require further purification, thereby reducing the application cost caused by the purification step.

[0033] 4. The chitosanase expressed by the recombinant Pichia pastoris engineered strain of the present invention is used to enzymatically hydrolyze colloidal chitosan. The obtained chitosan oligosaccharide has a single degree of polymerization and is chitotriose, which is expected to achieve large-scale preparation and subsequent application of chitotriose. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram showing the results of agarose gel electrophoresis detection of chitosanase gene amplification and expression vector construction in Examples 1 and 2 of the present application;

[0035] Figure 2 This is a schematic diagram of the construction of the chitosanase expression vector in Example 2 of the present application;

[0036] Figure 3 This is an SDS-PAGE electrophoresis diagram of the chitosanase expressed by the recombinant Pichia pastoris engineered strain in Example 3 of the present application;

[0037] Figure 4 This is a thin layer chromatography chromatogram of the chitosanase hydrolysis product expressed by the recombinant Pichia pastoris engineered strain in Example 4 of the present application. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0039] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0040] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0041] Example 1 Amplification of chitosanase gene Csn1 and filter Trans1 T1 / T1 Simple- Csn1 Positive strains

[0042] 1.1 The fungus Glomerella from the Anhui Provincial Key Laboratory of Modern Biomanufacturing Gongronella After sp. w5 bacteria were removed and activated, RNA was extracted and reverse transcribed to synthesize double-stranded cDNA.

[0043] 1.2 Design cloning Csn1The primers for the gene (the sequence of the upstream primer Csn1-F1 is shown in SEQ ID No: 3; the sequence of the downstream primer Csn1-R1 is shown in SEQ ID No: 4) were used to Gongronella sp. w5 RNA reverse transcription synthesized cDNA as a template to amplify the chitosanase gene Csn1 PCR amplification conditions reference Trans Taq ® -T DNA Polymerase instructions. Amplification system see Table 1, amplification conditions see Table 2.

[0044] SEQ ID No: 3: 5'-ATGAAGTCACTCACTTTGATTGC-3';

[0045] SEQ ID No: 4: 5'-TTACAGAGCAACAGAGCTATGG-3'.

[0046] Table 1 PCR amplification system (50 μL)

[0047]

[0048] Table 2 PCR amplification conditions

[0049]

[0050] 1.3 Ligate the recovered PCR product to the T1 Simple vector. Ligation system: Csn1 Recovered product: 4.5 μL; T1 Simple vector: 0.5 μL. Ligation conditions: 25 o C, 30 min.

[0051] 1.4 Transform the ligation product into Trans1 T1 competent cells to obtain transformants.

[0052] 1.5 Select the transformants from the previous step for colony PCR verification, transfer the transformants that have been successfully verified by PCR to LB medium, and incubate at 37 o C. Cultured in a shaker at 200 rpm for 14 h, the transformant plasmid was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing sequences were aligned to obtain Trans1 T1 / T1 Simple- Csn1 Positive strains.

[0053] Example 2 Construction of pPIC9K- Csn1 Expression vector and screening Pichia pastoris GS115 / pPIC9K- Csn1 Positive strains

[0054] 2.1 Extracting Trans1 T1 / T1 Simple- Csn1 Plasmids of positive strains.

[0055] 2.2 Design the signal peptide and introduce it into the upstream and downstream primers respectively Avr II Bln I) and Not I restriction enzyme site primers (the sequence of the upstream primer Csn1-F2 is shown in SEQ ID No: 5; the sequence of the downstream primer Csn1-R2 is shown in SEQ ID No: 6), with Trans1 T1 / T1 Simple- Csn1 The plasmid was used as a template for PCR amplification to increase the enzyme cutting site. Csn1 The product was recovered. PCR amplification conditions were based on the PrimeSTAR HS DNA Polymerase instructions. The amplification system is shown in Table 3, and the amplification conditions are shown in Table 4.

[0056] SEQ ID No: 5: 5'-ACCTAGGCAGAATAAGTACTGCAAGAATGAAT-3';

[0057] SEQ ID No: 6: 5'-ATATGCGGCCGCTTACAGAGCAACAGAGCTATGG-3'.

[0058] Table 3 PCR amplification system (50 μL)

[0059]

[0060] Table 4 PCR amplification conditions

[0061]

[0062] 2.3 The recovered PCR products and pPIC9K vector were double-digested ( Avr II and Not I restriction endonuclease), double enzyme digestion reaction system is shown in Table 5, 37 o C enzyme digestion for 90 min. After the digestion was completed, purification and recovery were performed respectively.

[0063] Table 5 Double enzyme digestion reaction system (50 μL)

[0064]

[0065] 2.4 Recover the enzyme digestion Csn1 The gene was ligated to the pPIC9K vector. Csn1 The molar ratio of the amount of the carrier is 5:1, and under the action of T4 DNA ligase, 22o C ligation was performed for 60 min, and the ligation product was transformed into Trans1 T1 competent cells to obtain transformants.

[0066] 2.5 The transformants were cultured in liquid LB medium overnight (37 o C, 200 rpm), extract the plasmid, perform double enzyme digestion verification, and obtain Trans1 T1 / pPIC9K- Csn1 Positive recombinant strains.

[0067] 2.6 Extraction of positive recombinant strain Trans1 T1 / pPIC9K- Csn1 plasmids and restriction enzymes Sac I linearized the plasmid under the following conditions: 37 o The reaction was continued at C for 90 min. The linearized recombinant plasmid was purified and recovered.

[0068] 2.7 The purified linearized recombinant plasmid was electroporated into Pichia pastoris GS115 competent cells. o After 2 h of recovery culture at C, yeast cells were collected by low-speed centrifugation and spread on MD medium plates. o C, culture for 2-3 days.

[0069] 2.8 Yeast cells grown on MD plates were cultured in BMGY liquid medium, and the yeast genome was extracted. PCR amplification was performed using the genome as a template and the universal primers α-FACTOR / 3'-AOX in place of the Csn1-F2 / Csn1-R2 primers in Table 3. The rest of the amplification system was the same as in Table 3. The extension time was changed to 1 min 30 s in the amplification conditions, and the rest was the same as in Table 4. The PCR products were subjected to agarose gel electrophoresis to verify whether the band size was consistent with the expectations. The products that met the expectations were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. Pichia pastoris GS115 / pPIC9K- Csn1 Positive strains.

[0070] Figure 1 The results of agarose gel electrophoresis detection of chitosanase gene amplification and expression vector construction in Examples 1 and 2 are shown in FIG; Figure 1 As can be seen in A, PCR amplification Csn1 The electrophoresis band positions of the genes were consistent with expectations; Figure 1 B and Figure 1 C are Csn1 The electrophoresis diagram after double enzyme digestion of the gene and pPIC9K shows that the band positions are consistent with expectations, and pPIC9K is cut into a linear plasmid; Figure 1 D is Trans1 T1 / pPIC9K- Csn1The plasmid double enzyme digestion electrophoresis of the strain showed that it was successfully inserted into the pPIC9K vector Csn1 Gene; Figure 1 E electrophoresis results showed that the positive Trans1 T1 / pPIC9K- Csn1 Plasmids of strains Sac I enzyme linearization; Figure 1 F electrophoresis diagram shows that Pichia pastoris Csn1 The transformant genome was extracted well; Figure 1 G is Pichia pastoris Csn1 The results of electrophoresis of PCR verification of transformant genome showed that the transformant was successfully isolated from Pichia pastoris. Csn1 Amplification in the transformant genome Csn1 Gene, Csn1 Successfully transformed into Pichia pastoris.

[0071] Figure 2 Schematic diagram of the chitosanase expression vector construction in Example 2 of this application. As shown in the figure, the chitosanase gene is designed to be inserted into the restriction site of the pPIC9K vector Avr II Bln I) and Not Between I.

[0072] Example 3 Inducible expression of chitosanase Csn1

[0073] 3.1 Recombinant Pichia pastoris engineered strain Pichia pastoris GS115 / pPIC9K- Csn1 Inoculate into 5 mL of BMGY medium, 28 o C, 200 rpm overnight activation culture.

[0074] 3.2 Transfer the inoculum to 50 mL of BMGY medium at 1% (v / v) for 28 o C, culture overnight at 200 rpm, and collect yeast cells by centrifugation.

[0075] 3.3 Transfer the yeast cells to 400 mL of BMM medium. OD 600 About 1.0, 28 o C, 200 rpm for 7 d (while the culture medium was supplemented with 0.5% (v / v) methanol every 24 h during 7 d to induce chitosanase production).

[0076] 3.4 Cultivate based on 4 oC. Centrifuge at 8000 rpm for 30 min to remove yeast cells, collect the supernatant crude enzyme solution, filter the crude enzyme solution through a 0.45 μm microporous filter membrane, and then use a tangential flow ultrafiltration instrument to concentrate it by ultrafiltration. Then, adjust the pH to 6.0 using 20 mM sodium acetate buffer.

[0077] 3.5 The expression and purity of chitosanase were detected by SDS-PAGE.

[0078] 3.6 The chitosanase hydrolysis activity was detected using 1% (w / v) colloidal chitosan as substrate.

[0079] Figure 3 This is the SDS-PAGE electrophoresis diagram of chitosanase expressed by the recombinant Pichia pastoris engineered strain in Example 3. As can be seen from the figure, the chitosanase expressed by the recombinant Pichia pastoris engineered strain of the present invention has high purity and does not require further purification.

[0080] Example 4 Application of recombinant chitosanase in the preparation of chitosan oligosaccharides

[0081] 100 μL (about 140 μg) of chitosanase solution prepared in Example 3 was added to 400 μL of 1.0% (w / v) colloidal chitosan (pH 6.0, deacetylation degree 90%-95%) and placed in a 50 o The reaction was carried out at 400 °C for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. The system was boiled for 10 min to inactivate the enzyme, and centrifuged at 8000 g for 10 min. The supernatant was the enzymatic hydrolysis product.

[0082] The samples were spotted in the order of 30 mg / mL chitosan oligosaccharide mixed standard (2 μL), enzymatic hydrolysates obtained at different reaction times (4 μL), and enzyme solution (2 μL), with a spot spacing of 8 mm. After spotting, the silica gel plate was completely blown dry and developed with n-butanol:isopropanol:ammonia water (3:6:4, v:v:v) as the developing agent, with a spot spacing of 8 cm. After developing, the silica gel plate was blown dry, immersed in 0.1% ninhydrin ethanol solution (w / v) for staining for 5 s, removed and immediately blown dry, and then placed on an electric stove for heating and color development.

[0083] The results of thin layer chromatography are as follows Figure 4 As shown in the figure, within 0.5 h to 24 h, excess chitosanase Csn1 hydrolyzed 1.0% colloidal chitosan to produce chitotriose, a single-degree-of-polymerization product. The amount of product produced gradually increased with reaction time. This enzyme can be used to produce chitotriose.

[0084] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A chitosanase, characterized in that Its amino acid sequence is shown in SEQ ID No:

1.

2. A gene encoding the chitosanase according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID No:

2.

3. A recombinant expression vector containing the gene of the chitosanase according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pPIC9K- Csn1 Expression vector.

5. A recombinant expression strain containing the recombinant expression vector according to claim 3.

6. The recombinant expression strain according to claim 5, characterized in that The recombinant expression strain is a recombinant Pichia pastoris engineered strain obtained by transferring the recombinant expression vector according to claim 3 into Pichia pastoris. Pichia pastoris GS115 / pPIC9K- Csn1 The recombinant Pichia pastoris engineered strain was deposited in the China Center for Type Culture Collection on July 14, 2022, with the deposit number CCTCC NO. M 20221107.

7. A method for preparing the chitosanase according to claim 1, characterized in that: The recombinant expression strain according to claim 6 is cultured in a culture medium, and the protein having chitosanase activity is collected.

8. Use of the chitosanase according to claim 1 in preparing chitosan oligosaccharides.

9. The use according to claim 8, characterized in that Chitosan oligosaccharides are obtained by reacting chitosanase with a 0.25% to 1.5% (w / v) colloidal chitosan solution as a substrate for more than 0.5 h.

10. The use according to claim 9, characterized in that The reaction conditions are: temperature 30-70° C., pH 3.5-6.0.

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