Preparation method and application of chitosanase BNCSN

Abstract

The invention discloses a preparation method of chitosanase BNCSN and an application of the chitosanase BNCSN. According to the invention, a potential chitosanase gene derived from bacillus nakamurai is subjected to secretory expression in pichia pastoris, and the potential chitosanase gene is derived from bacillus nakamurai. The chitosanase obtained by the invention has higher hydrolytic activity to a chitosan substrate, a crude enzyme liquid generated by shake flask fermentation has the capability of degrading 5g of chitosan by 1mL (the content of crude protein is about 0.60 mg), while about 150mg of non-specific commercial neutral protease is needed for hydrolyzing the same amount of chitosan, the enzymolysis efficiency is improved by more than 200 times, and the yield is increased by more than 100%. The industrial application potential of large-scale preparation of chitosan oligosaccharide is realized.

Description

Technical Field

[0001] The invention relates to a preparation method and application, and in particular to a preparation method and application of chitosanase BNCSN. Background Art

[0002] Chitosan oligosaccharides are oligosaccharides composed of 2-10 aminoglucosides connected by β-1,6-glycosidic bonds. They have the characteristics of good water solubility and high biological activity. In the industrial field, chitosan oligosaccharides can be used as food additives for preservation and seasoning; in cosmetics, they can play a role in moisturizing and antibacterial; they can also be used in the textile industry to improve the antibacterial and wrinkle resistance of fabrics. At present, the preparation methods of chitosan oligosaccharides mainly include chemical methods, which use acid and alkali reagents to degrade chitosan, but the product is not uniform; enzymatic methods, which use chitosanase to hydrolyze chitosan, and the product has high activity and good safety; physical methods, such as microwave and ultrasound assisted degradation, are simple to operate and environmentally friendly, but the efficiency may be low.

[0003] However, due to the lack of efficient and specific chitosanase in the enzymatic hydrolysis method, non-specific commercial enzymes containing chitosan hydrolysis activity, such as cellulase, protease and lipase, are usually used in the enzymatic preparation. These commercial enzymes have low hydrolysis activity, and it is necessary to develop chitosanase with higher hydrolysis activity for efficient industrial preparation of chitosan oligosaccharides. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for preparing a specific chitosanase, chitosanase BNCSN, which is economical and efficient.

[0005] To achieve the above object, the technical solution of the present invention is as follows: A method for preparing chitosanase BNCSN is carried out according to the following steps: S10, codon optimization of the potential chitosanase gene from Bacillus Nakamura as shown in the gene sequence SEQ ID NO.1 is performed according to the preference of Pichia pastoris codon usage, and the optimized chitosanase gene sequence is shown in SEQ ID NO.2; S11, the optimized chitosanase gene is fully synthesized, and the synthesized gene sequence is recorded as bncsn; S12, constructing the synthesized chitosanase gene bncsn into an expression vector to obtain a recombinant vector; S13, introducing the chitosanase gene bncsn treated in step S12 into Pichia pastoris cells; S14, inducing the Pichia pastoris cells treated in step S13 and obtaining the chitosanase BNCSN solution secreted and expressed by the cells.

[0006] Furthermore, in step S10, while codon optimization is being performed on the potential chitosanase gene as shown in the gene sequence SEQ ID NO.1, the signal peptide needs to be removed.

[0007] Furthermore, the detailed operation steps of step S12 are as follows: S12.a, using endonuclease to obtain the target gene fragment of the chitosanase gene bncsn cloning vector; S12.b, use endonucleases to obtain large fragments of the expression vector; S12.c, connect the gene fragments obtained in S12.a and S12.b to obtain a recombinant vector.

[0008] Furthermore, in both steps S12.a and S12.b, restriction endonucleases Xho I and Not I are used to perform double enzyme digestion on the bncsn cloning vector and expression vector containing the chitosanase gene; the expression vector in step S12.b is pPIC9; and the recombinant vector in step S12.c is a recombinant plasmid.

[0009] Furthermore, between steps S12 and S13, in order to confirm that the target chitosanase gene bncsn has been constructed into the expression vector, the recombinant vector is double-digested with Xho I and Not I, and the product is subjected to agarose gel electrophoresis.

[0010] Furthermore, the detailed operation steps of step S13 are as follows: S13.a, linearizing the recombinant vector with restriction endonuclease BglII, separating and cutting out the large nucleic acid fragment containing the target gene by gel electrophoresis; S13.b, electroporation into Pichia pastoris; S13.c, recombinant colonies were obtained by screening on histidine auxotrophic MD plates.

[0011] Furthermore, the detailed operation steps of step S14 are as follows: S14.a, select several recombinant colonies in S13.c and perform shake flask induction expression; S14.b, after the induction expression was completed, the crude enzyme supernatant was obtained by centrifugation; S14.c, using the obtained crude enzyme supernatant to hydrolyze 1% chitosan substrate, the strain with the highest expression level was screened by viscosity method for the preparation of chitosanase BNCSN.

[0012] The beneficial effects of the present invention are as follows: first, the chitosanase gene of the present invention is derived from Bacillus nakamurai and is secreted and expressed using a Pichia pastoris expression system. The Pichia pastoris expression system has been used to express food-grade enzyme preparations such as lactase and phospholipase C, and it itself has also been used to produce xylanase (GB2760-2014). Therefore, the chitosanase gene derived from Bacillus nakamurai is secreted and expressed in Pichia pastoris, and the product chitosanase can become an enzyme preparation for the production of food-grade chitooligosaccharides.

[0013] Secondly, integrating the target gene into the Pichia pastoris genome through genetic recombination is more stable than the expression of free plasmid vectors and is not easily lost due to multiple subcultures. Enzymes secreted and expressed in Pichia pastoris do not require cell disruption or other processes because they are secreted extracellularly. The crude enzyme solution obtained from the fermentation broth can be directly used for the preparation of chitosan oligosaccharides without purification.

[0014] In addition, during the gene expression process, the signal peptide of the Pichia vector is used to achieve secretory expression, and the signal peptide sequence of chitosanase itself is removed, which brings many beneficial effects. The signal peptide of the Pichia vector is compatible with the secretion system of Pichia, can accurately guide the protein into the secretion pathway, has strong versatility, and does not need to be optimized separately for the signal peptide of chitosanase itself, reducing the complexity of the experiment; removing the signal peptide of chitosanase itself can avoid potential conflicts with the secretion system of Pichia, reduce signal recognition errors, and make the regulation of the secretion process simpler and clearer. This helps chitosanase to fold and process efficiently and correctly in the endoplasmic reticulum of Pichia, improve its activity and stability, ensure the accuracy of the signal peptide cleavage site, and generate chitosanase with the correct amino acid sequence and structure. In addition, it can also make chitosanase more effectively secreted into the extracellular medium, facilitate subsequent separation and purification, improve product purity, reduce the risk of degradation by proteases in cells, and ultimately improve the yield and quality of the target protein.

[0015] Finally, the chitosanase gene of the present invention is optimized according to the codon preference of Pichia pastoris, so that efficient secretory expression can be achieved in Pichia pastoris.

[0016] It should be noted that the chitosanase BNCSN in the present invention can be used alone to degrade chitosan to prepare chitooligosaccharides; or the chitosanase BNCSN can be used in combination with other chitosan-degrading enzymes to synergistically degrade chitosan.

[0017] The present invention also provides an application of preparing chitosan oligosaccharides by chitosanase BNCSN, which is carried out according to the following steps: S20, elution of chitosan; S21, placing the chitosan treated in step S20 into a solution to fully dissolve and fix the volume; S22, adding the prepared chitosanase BNCSN solution to the solution treated in step S21, and stirring to react; S23, after a certain stirring reaction time in S22, the insoluble matter is removed by centrifugation, and the supernatant is freeze-dried to obtain the finished chitosan oligosaccharide.

[0018] Furthermore, the elution procedure in step S20 is as follows: the acetonitrile concentration is decreased from 70% to 50%; the concentration of 0.1 M ammonium formate at pH 3.2 is increased from 30% to 50%; and the elution time is 40 min.

[0019] The chitosan substrate was hydrolyzed by using the crude enzyme supernatant of the induced expressed chitosanase BNCSN and the polymerization degree and composition of the product were analyzed by liquid phase method. The analysis results showed that the obtained crude enzyme solution of chitosanase BNCSN could hydrolyze chitosan. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The recombinant expression vector bncsn-pPIC9 in the embodiment of the present invention and the gel electrophoresis pattern of its Xho I and Not I double enzyme digestion products; Figure 2 The SDS-PAGE pattern of the supernatant of the fermentation broth of Pichia pastoris engineered bacteria containing the chitosanase gene in the embodiment of the present invention; Figure 3 The HPLC analysis chart of the enzymatic hydrolysis product chitosan oligosaccharide COS-90-BNCSN in the example of the present invention. DETAILED DESCRIPTION

[0021] It should be noted that the reagents and biological materials used below are all commercial products unless otherwise specified. If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. In this example, Pichia pastoris GS115 was used.

[0022] The present invention provides a method for preparing chitosanase BNCSN, which is carried out according to the following steps: S10, according to the preference of Pichia pastoris codon usage, the potential chitosanase gene from Bacillus Nakamura as shown in the gene sequence SEQ ID NO.1 was codon optimized and the signal peptide was removed. The optimized chitosanase gene sequence is shown in SEQ ID NO.2. The identity of the optimized nucleic acid sequence to the original sequence is 74%.

[0023] S11, the optimized chitosanase gene is fully synthesized, and the synthesized gene sequence is recorded as bncsn.

[0024] S12, constructing the synthesized chitosanase gene bncsn into an expression vector to obtain a recombinant vector; The specific operation steps of S12 are as follows: S12.a, double-digest the cloning vector containing the chitosanase gene bncsn using restriction endonucleases Xho I and Not I to obtain the target gene fragment; S12.b, double digest the expression vector pPIC9 with the same endonuclease to recover the large fragment; S12.c, connect the gene fragments obtained in S12.a and S12.b to obtain a recombinant vector, i.e., a recombinant plasmid, named bncsn-pPIC9.

[0025] Before proceeding to the next step, in order to confirm that the target chitosanase gene bncsn has been constructed into the expression vector, the recombinant plasmid was double-digested with Xho I and Not I, and the product was subjected to agarose gel electrophoresis. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that after double enzyme digestion, the target gene fragment appeared between 750 bp and 1000 bp, which is consistent with the fragment length of bncsn 779 bp.

[0026] S13, introducing the chitosanase gene bncsn treated in step S12 into Pichia pastoris cells; The specific operation steps of S13 are as follows: S13.a, linearizing the obtained recombinant plasmid bncsn-pPIC9 with restriction endonuclease BglII, separating and cutting out the large nucleic acid fragment containing the target gene by gel electrophoresis; S13.b, electroporation into Pichia pastoris; S13.c, recombinant colonies were obtained by screening on histidine auxotrophic MD plates.

[0027] S14, inducing the Pichia pastoris cells treated in step S13 and obtaining the chitosanase BNCSN solution secreted and expressed by the cells.

[0028] S14 The specific operation steps are as follows: S14.a, select several recombinant colonies from S13.c and perform shake flask induction expression: inoculate the colonies into 50 mL BMGY medium, culture at 28°C and 220 rpm for 48 h, add methanol to a final concentration of 1% for induction, and then add methanol every 24 h for a total of 72 h; S14.b, after the induction expression was completed, the crude enzyme supernatant was obtained by centrifugation; S14.c, using the obtained crude enzyme supernatant to hydrolyze 1% chitosan substrate, the strain with the highest expression level was screened by viscosity method for the preparation of chitosanase BNCSN.

[0029] SDS-PAGE was used to detect the protein expression of the strain with the highest activity. The results are as follows: Figure 2 As shown, a band appeared near 30 kDa, which was larger than the expected relative molecular mass of chitosanase (28.3 kDa), and it was speculated that glycosylation modification occurred. The protein concentration in the fermentation supernatant was 0.60 mg / mL as determined by the Bradford method. The above-mentioned MD agar plate, BMMY agar plate, BMGY medium, and BMMY medium are commonly used culture media for yeast expression systems and can be purchased directly or prepared according to existing literature techniques.

[0030] The embodiment of the present invention also provides an application of preparing chitosan oligosaccharides by chitosanase BNCSN, which is implemented according to the following steps: S20, chitosan was eluted. The elution procedure was as follows: the acetonitrile concentration was decreased from 70% to 50%; the concentration of 0.1 M and pH 3.2 formate was increased from 30% to 50%; the elution time was 40 min.

[0031] S21, weigh 1 g of the chitosan treated in step S20, add 10 mL of water and 0.3 mL of acetic acid, stir to fully dissolve it, and then add water to make the volume to 100 mL; S22, adding 0.2 mL of the prepared chitosanase BNCSN crude enzyme solution to the solution treated in step S21, and stirring the reaction at 40° C. for 48 hours; S23, after the stirring reaction is completed, the insoluble matter is removed by centrifugation, and the supernatant is freeze-dried to obtain the finished chitosan oligosaccharide, which is recorded as COS-90-BNCSN.

[0032] Weigh a certain amount of prepared freeze-dried chitosan oligosaccharide sample, prepare an acetonitrile aqueous solution (acetonitrile: water, 1:1, v / v) with a chitosan oligosaccharide concentration of 5 mg / mL, and filter it for high-performance liquid chromatography analysis. The high-performance liquid chromatograph is connected to an evaporative light scattering detector for oligosaccharide signal detection, and the chitosan oligosaccharide is separated using an XAmide column. The column temperature is 40°C, the flow rate is 1 mL / min, and the mobile phase is 0.1M formic acid ammonium (pH 3.2) and acetonitrile. The results are shown in Figure 3 As shown, chitosan oligosaccharides with a degree of polymerization mainly of 2-5 were obtained.

[0033] Combined with the test data of step S14.3, it can be seen that the protein concentration of the crude chitosanase BNCSN enzyme supernatant obtained by the preparation method of chitosanase BNCSN in this embodiment is 0.60 mg / mL. And in the application steps S20-S23 of preparing chitosan oligosaccharides by chitosanase BNCSN in this embodiment, it can be seen that under the disclosed reaction time and reaction conditions, the crude chitosanase BNCSN enzyme supernatant required to degrade 1g of chitosan is 0.2mL.

[0034] Therefore, it can be inferred that the chitosanase obtained by the present invention has a high hydrolysis activity on chitosan substrates. The crude enzyme solution produced by shake flask fermentation has the hydrolysis ability of 1 mL of crude enzyme solution (protein content of about 0.60 mg) to degrade 5 g of chitosan, while the degradation of the same amount of chitosan requires about 150 mg of commercial neutral protease, which theoretically improves the efficiency by more than 200 times. Therefore, the secretory chitosanase expressed by the present invention has the potential to replace the existing commercial enzymes for large-scale preparation of chitosan oligosaccharides, and has good industrial application prospects.

[0035] The above embodiment is only one of the preferred specific embodiments of the present invention. Common changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention are all included in the protection scope of the present invention.

Claims

1. A method for preparing chitosanase BNCSN, comprising the steps of: S10, codon optimization of the potential chitosanase gene from Bacillus Nakamura as shown in the gene sequence SEQ ID NO.1 is performed according to the preference of Pichia pastoris codon usage, and the optimized chitosanase gene sequence is shown in SEQ ID NO.2; S11, the optimized chitosanase gene is fully synthesized, and the synthesized gene sequence is recorded as bncsn; S12, constructing the synthesized chitosanase gene bncsn into an expression vector to obtain a recombinant vector; S13, introducing the chitosanase gene bncsn treated in step S12 into Pichia pastoris cells; S14, inducing the Pichia pastoris cells treated in step S13 and obtaining the chitosanase BNCSN solution secreted and expressed by the cells.

2. The method for preparing chitosanase BNCSN according to claim 1, characterized in that: In the step S10, while the codons of the potential chitosanase gene shown in the gene sequence SEQ ID NO.1 are being optimized, the signal peptide thereof needs to be removed.

3. The method for preparing chitosanase BNCSN according to claim 1, characterized in that: The detailed operation steps of step S12 are as follows: S12.a, using endonuclease to obtain the target gene fragment of the chitosanase gene bncsn cloning vector; S12.b, use endonucleases to obtain large fragments of the expression vector; S12.c, connect the gene fragments obtained in S12.a and S12.b to obtain a recombinant vector.

4. The method for preparing chitosanase BNCSN according to claim 3, characterized in that: In both steps S12.a and S12.b, restriction endonucleases Xho I and Not I are used to perform double enzyme digestion on the bncsn cloning vector and expression vector containing the chitosanase gene; the expression vector in step S12.b is pPIC9; and the recombinant vector in step S12.c is a recombinant plasmid.

5. The method for preparing chitosanase BNCSN according to claim 1, characterized in that: Between steps S12 and S13, in order to confirm that the target chitosanase gene bncsn has been constructed into the expression vector, the recombinant vector is double-digested with Xho I and Not I, and the product is subjected to agarose gel electrophoresis.

6. The method for preparing chitosanase BNCSN according to claim 1, characterized in that: The detailed operation steps of step S13 are as follows: S13.a, linearizing the recombinant vector with restriction endonuclease BglII, separating and cutting out the large nucleic acid fragment containing the target gene by gel electrophoresis; S13.b, electroporation into Pichia pastoris; S13.c, recombinant colonies were obtained by screening on histidine auxotrophic MD plates.

7. The method for preparing chitosanase BNCSN according to claim 6, characterized in that: The detailed operation steps of step S14 are as follows: S14.a, select several recombinant colonies in S13.c and perform shake flask induction expression; S14.b, after the induction expression was completed, the crude enzyme supernatant was obtained by centrifugation; S14.c, using the obtained crude enzyme supernatant to hydrolyze 1% chitosan substrate, the strain with the highest expression level was screened by viscosity method for the preparation of chitosanase BNCSN.

8. Use of the chitosanase BNCSN obtained according to any one of claims 1 to 7 to prepare chitosan oligosaccharides, according to the following steps: S20, elution of chitosan; S21, placing the chitosan treated in step S20 into a solution to fully dissolve and fix the volume; S22, adding the prepared chitosanase BNCSN solution to the solution treated in step S21, and stirring the solution for reaction; S23, after a certain stirring reaction time in S22, centrifuging to remove insoluble matter, and freeze-drying the supernatant to obtain the finished chitosan oligosaccharide.

9. The use of chitosanase BNCSN for preparing chitosan oligosaccharides according to claim 8, characterized in that: The elution procedure in step S20 is as follows: the acetonitrile concentration is decreased from 70% to 50%; the concentration of 0.1 M ammonium formate at pH 3.2 is increased from 30% to 50%; and the elution time is 40 min.

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