阅读说明：本技术 一种几丁质酶、AfChi18基因及其表达方法与应用 (Chitinase and AfChi18 gene as well as expression method and application thereof ) 是由 杨登峰 伍雅励 潘丽霞 阳丽艳 李红亮 于 2021-10-13 设计创作，主要内容包括：本发明提供了一种几丁质酶、AfChi18基因及其表达方法与应用,属于基因工程技术领域。本发明提供的几丁质酶的氨基酸序列如SEQ ID No.1所示,编码所述几丁质酶的AfChi18基因的核苷酸序列如SEQ ID No.2所示,本发明还提供了包含AfChi18基因的重组质粒以及用于表达所述重组质粒的重组菌株。本发明提供的几丁质酶为典型的几丁质内切酶,大小为40KDa,该几丁质酶的最适反应温度为45℃,在20℃～50℃范围内热稳定性好；最适pH为5.0,在pH4.0～6.0具有较高的酶活,表明本发明提供的几丁质酶具有较高的稳定性。(The invention provides chitinase and AfChi18 genes and an expression method and application thereof, belonging to the technical field of genetic engineering. The amino acid sequence of the chitinase is shown as SEQ ID No.1, the nucleotide sequence of the AfChi18 gene for coding the chitinase is shown as SEQ ID No.2, the invention also provides a recombinant plasmid containing the AfChi18 gene and a recombinant strain for expressing the recombinant plasmid. The chitinase provided by the invention is a typical chitinase endonuclease, the size of the chitinase is 40KDa, the optimal reaction temperature of the chitinase is 45 ℃, and the thermal stability is good within the range of 20-50 ℃; the optimum pH value is 5.0, and the chitinase has higher enzyme activity at the pH value of 4.0-6.0, which shows that the chitinase provided by the invention has higher stability.)

1. The chitinase is characterized in that the amino acid sequence of the chitinase is shown as SEQ ID No. 1.

2. An AfChi18 gene encoding the chitinase of claim 1, wherein the AfChi18 gene has a nucleotide sequence shown in SEQ ID No. 2.

3. A recombinant plasmid comprising a nucleotide sequence of AfChi18 gene encoding the chitinase of claim 1.

4. A recombinant strain expressing the recombinant plasmid of claim 3.

5. A method for expressing chitinase according to claim 1, comprising the steps of:

(1) connecting the AfChi18 gene with a plasmid vector to obtain a recombinant plasmid;

(2) transforming the recombinant plasmid into a clone strain to obtain a recombinant clone strain;

(3) extracting plasmids in the recombinant clone strains, and transforming the plasmids into expression strains to obtain recombinant expression strains;

(4) and (3) carrying out induction culture on the recombinant expression strain, collecting and crushing thalli, and obtaining the chitinase.

6. The expression method of claim 5, wherein the AfChi18 gene amplification primers are shown as SEQ ID No.3 and SEQ ID No. 4.

7. The expression method of claim 5 or 6, wherein the plasmid vector is pET28a, the cloning strain is E.coli DH5 α, and the expression strain is E.coli BL21DE₃codonplus RIL。

8. The expression method according to claim 7, wherein the induction culture in the step (5) is performed by inoculating the recombinant expression strain into a first LB medium containing kanamycin, culturing at 36-38 ℃ for 12-18 hours, inoculating the recombinant expression strain into a second LB medium containing kanamycin at an inoculum size of 1% v/v, and culturing at 36-38 ℃ to OD₆₀₀And (3) adding isopropyl-beta-D-thiogalactoside, and inducing at 25-30 ℃ and 180-200 rpm for 8-10 h.

9. The expression method according to claim 8, wherein the concentration of kanamycin in the first LB medium and the second LB medium is independently 48 to 52 mg/ml; the final concentration of the isopropyl-beta-D-thiogalactoside in the second LB culture medium is 0.0008-0.0012M.

10. Use of the chitinase of claim 1 for degrading chitin.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to chitinase and AfChi18 genes as well as an expression method and application thereof.

Background

Chitin, also called chitin, has a molecular formula of [ C₈H₁₃O₅N]_n. The chitin is a structural homopolysaccharide formed by polymerizing N-acetylglucosamine (N-acetyl-D-glcnac-glucosamine) through beta-1, 4 glycosidic bonds, widely exists in shells of crustaceans, shells of insects and cell walls of fungi, has the chitin content of about 25 percent in shrimp shells, and has low cost and high yield for extracting the chitin from the shrimp shells. Chitin, an important biological resource, is second only to cellulose in earth, and has a natural annual production of 10¹¹Ton, a huge renewable resource. In recent years, green application of chitin has become a research hotspot.

The degradation product of chitin is chitin oligosaccharide, chitin monosaccharide and their corresponding derivatives with high added value, has the functions of resisting microorganism, resisting oxidation, gene therapy, immune cell proliferation and tumor growth inhibition, and can be widely used in feed and food as additive. The chitin oligosaccharide is prepared by chemical extraction and acetylation. The chemical extraction method generally uses an acid hydrolysis method, which utilizes strong acid to degrade chitin into glucosamine, and obtains N-acetylglucosamine after acetylation; or directly degrading chitin with diluted acid to generate chitin oligosaccharide. The chemical extraction method has the advantages of violent reaction, high cost, serious environmental pollution and low polymerization degree of chitin oligosaccharide products. In order to meet the national green sustainable development policy, the green chitin degradation by the biological enzyme method is a research hotspot. The chitin oligosaccharide prepared by degrading chitin by the biological enzyme method has the advantages of mild reaction process, good biological activity and high-efficiency utilization of waste chitin resources.

Chitinases are classified into endo-chitinases, exo-chitinases and N-acetylglucosidases according to the cleavage mode of glycosidic bonds. Endo-chitinase acts randomly on any of the glycosidic bonds of the long chain chitin and hydrolyzes it into chitin oligosaccharides. Exochitinase, unlike endo-chitinase, degrades chitin from its non-reducing ends into (GlcNAc)₂. However, N-acetylglucosidases do not act directly on chitin, but only hydrolyze chitin oligosaccharides to GlcNAc. Chitinase degrades waste chitin to generate chitin oligosaccharide, the reaction condition is mild, the chitin oligosaccharide is green and environment-friendly, and the raw materials have low cost and high yield, thereby completely meeting the requirements of industrial production.

Therefore, the method has important significance in digging novel chitinase with high enzyme activity and efficiently utilizing biomass polysaccharide.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide chitinase and AfChi18 genes, and an expression method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides chitinase, and the amino acid sequence of the chitinase is shown as SEQ ID No. 1.

The invention also provides an AfChi18 gene for coding the chitinase, and the nucleotide sequence of the AfChi18 gene is shown as SEQ ID No. 2.

The invention also provides a recombinant plasmid which comprises a nucleotide sequence of the AfChi18 gene for coding the chitinase.

The invention also provides a recombinant strain which expresses the recombinant plasmid.

The invention also provides an expression method of the chitinase, which comprises the following steps:

(1) connecting the AfChi18 gene with a plasmid vector to obtain a recombinant plasmid;

(2) transforming the recombinant plasmid into a clone strain to obtain a recombinant clone strain;

(3) extracting plasmids in the recombinant clone strains, and transforming the plasmids into expression strains to obtain recombinant expression strains;

(4) and (3) carrying out induction culture on the recombinant expression strain, collecting and crushing thalli, and obtaining the chitinase.

Preferably, the AfChi18 gene amplification primers are shown as SEQ ID No.3 and SEQ ID No. 4.

Preferably, the plasmid vector is pET28a, the cloning strain is E.coli DH5 alpha, and the expression strain is E.coli BL21DE₃ codonplus RIL。

Preferably, the induction culture in step (5) is performed by inoculating the recombinant expression strain into a first LB medium containing kanamycin, culturing at 36-38 ℃ for 12-18 h, inoculating the recombinant expression strain into a second LB medium containing kanamycin at an inoculum size of 1% v/v, and culturing at 36-38 ℃ to OD₆₀₀And (3) adding isopropyl-beta-D-thiogalactoside, and inducing at 25-30 ℃ and 180-200 rpm for 8-10 h.

Preferably, the concentration of kanamycin in the first LB culture medium and the second LB culture medium is 48-52 mg/ml independently; the final concentration of the isopropyl-beta-D-thiogalactoside in the second LB culture medium is 0.0008-0.0012M.

The invention also provides application of the chitinase in degrading chitin.

The product of the chitinase enzymolysis colloidal chitin provided by the invention is mainly (GlcNAc)₂And (GlcNAc)₃With a small amount of GlcNAc, typical of endoplasmic enzymes, of size 40 KDa. The optimum reaction temperature of the chitinase is 45 ℃, and the thermal stability is good within the range of 20-50 ℃; the optimum pH value is 5.0, and the chitinase has higher enzyme activity at the pH value of 4.0-6.0, which shows that the chitinase provided by the invention has higher stability. The influence test of metal ions and chemical reagents shows that Co²⁺、Na⁺、Mg^{2 +}、NH₄ ⁺、Ca²⁺、Zn²⁺、K⁺Tris, urea, and Tris internally cutting the chitinThe enzyme has an activating effect; cu²⁺、Fe³⁺、Mn²⁺、Ba²⁺SDS and EDTA have inhibitory effect on chitinase.

Drawings

FIG. 1 shows the results of SDS-PAGE detection of chitinase expressed in example 2; in the figure, M: color pre-stained protein markers, 1: total broken cell protein before centrifugation (final concentration 4.5M), 2: chitin crude enzyme solution, 3: broken cell total protein is protein grafted in the Ni-NTABeads 6FF column loading process, 4: heteroprotein of total broken cell protein eluted with 100ml imidazole at 50mM concentration, 5: 4, eluting the hybrid protein in the solution with 100ml of imidazole with the concentration of 500mM to obtain purified protein;

FIG. 2 is a standard curve for N-acetylglucosamine;

FIG. 3 is a temperature profile of the enzymatic activity of chitinase in example 4 of the present invention;

FIG. 4 is a graph showing the temperature stability of the enzymatic activity of chitinase in example 4 of the present invention;

FIG. 5 is a pH curve of the enzymatic activity of chitinase in example 4 of the present invention;

FIG. 6 is a graph showing the pH stability of the enzymatic activity of chitinase in example 4 of the present invention;

FIG. 7 shows the effect of metal ions and chemical reagents on the enzymatic activity of chitinase in example 4 of the present invention.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

EXAMPLE 1 construction of recombinant plasmid pET28a-Afchi18

Step 1, designing PCR amplification primer

AfChi18-F-5’-GTGCCGCGCGGCAGCCATATGATGTTCTCCGGATCCATCTTCC(SEQ ID No.3)

AfChi18-R-5’-CTCGAGTGCGGCCGCAAGCTTTCACATATCATGCAAGGTCTTATACCC(SEQ ID No.4)

Step 2, amplification of AfChi18 Gene Using the primers of step 1

The nucleotide sequence of the AfChi18 gene is taken as a template (shown as SEQ ID No. 2), and the AfChi18 gene is amplified by adopting a PCR method.

The reaction system of PCR amplification is as follows: template 0.5 μ l; 0.3. mu.l of the forward primer (SEQ ID No. 3); 0.3. mu.l of downstream primer (SEQ ID No. 4); 2 × Taq Master Mix 10 μ l; ddH₂O8.9 μ l; the total volume of the reaction system was 20. mu.l.

The reaction conditions for PCR amplification are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 20 s; extension at 72 ℃ for 2min for 35 cycles; after 72 ℃ extension, 10min and finally 16 ℃ heat preservation.

And 3, recovering the purified amplification product after PCR amplification is finished to obtain a purified DNA product.

In step 4, the vector pET28a was digested with restriction enzymes Nde I and Hind III, and the product was recovered to obtain the purified vector pET28a (5304 bp).

And 5, carrying out double digestion on the amplification product obtained in the step 3 by using the same restriction enzymes Nde I and Hind III, and carrying out homologous recombination and ligation (by adopting a PCR method) on the product subjected to double digestion and purification in the step 4 by using 2 XTaq PCR Master Mix DNA ligase.

The PCR homologous recombination ligation reaction system is as follows: 1. mu.l of purified DNA product; carrying out enzyme digestion on the purified vector pET28a3 mu l;Mix 5μl；ddH₂o1 mu l; the total volume of the reaction system was 10. mu.l.

The reaction procedure of PCR homologous recombination ligation is as follows: the homologous recombination ligation reaction system is mixed evenly, placed in a water bath kettle at 50 ℃ for reaction for 5min, and then immediately placed on ice (0 ℃) for cooling. The reaction mixture was purified by the GeneJET PCR purification kit (thermo scientific) to obtain the product recombinant plasmid pET28a-Afchi 18.

Example 2 expression of chitinase Afchi18 Gene

Step 1, constructing a recombinant clone strain pET28a-AfChi18-E

The recombinant plasmid pET28a-Afchi18 was chemically transformed to E.coli DH5a, and the cloned strain pET28a-AfChi18-E.coli DH5 alpha was obtained by colony PCR screening and sequencing.

Step 2, constructing a recombinant expression strain pET28a-AfChi18-E₃ codon plus RIL

Extracting recombinant Plasmid from recombinant clone strain pET28a-Afchi18-E.coli DH5a by TIANPrep Mini Plasmid Kit (TIANGEN), and chemically converting to expression strain E.coli BL21DE₃In the codonplus RIL, a single colony is picked and directly cultured in an LB culture medium at 37 ℃ and 220rpm overnight to obtain a recombinant expression strain pET28a-Afchi18-E₃ codon plus RIL。

Step 3, expression of recombinant expression strains

The recombinant expression strain pET28a-Afchi18-E.coli BL21DE₃codon plus RIL was inoculated into 10ml of LB medium containing kanamycin (50mg/ml), cultured overnight at 37 ℃ for 16 hours, inoculated into 500ml of LB medium containing kanamycin (50mg/ml) in an inoculum size of 1% v/v, and cultured at 37 ℃ to OD₆₀₀isopropyl-beta-D-thiogalactoside (IPTG) was added to a final concentration of 0.001M in LB medium at 25-30 ℃ for 10h at 180-200 rpm ═ 0.7.

Step 4, collecting and crushing the thalli to obtain crude chitinase enzyme liquid

After the expression is finished, centrifuging the bacteria liquid subjected to induced culture at 4 ℃ and 7400rpm for 25min, collecting thalli, resuspending the thalli by using a citric acid-sodium dihydrogen phosphate buffer solution, and ultrasonically crushing the thalli on ice at an interval of 2s for 1s every time for 20 min; then centrifuging at 1200rpm for 25min at 4 ℃ and collecting the supernatant to obtain the crude chitinase solution.

Step 5, purifying chitin crude enzyme liquid

And (3) performing protein purification on the crude chitinase solution obtained in the step (4) by using a His-TAG column, and performing SDS-PAGE on the purified recombinant protein solution (namely, chitinase) to obtain a protein band of about 40kDa (shown in figure 1).

EXAMPLE 3 enzymatic Properties of chitinase

Enzyme activity determination

A calibration curve was prepared by the potassium ferricyanide method (Schales method).

And (3) drawing an acetylglucosamine standard curve: accurately weighing a certain amount ofN-acetylglucosamine (N-GlcNAc), soluble in ddH₂O, and formulated as a standard reagent at a final concentration of 10 mM. N-acetylglucosamine standard reagent (10mM N-GlcNAc), ddH were added in the order of Table 1₂O, Schales reagent, mixed well to obtain a series of standard solutions (each set is 3 times repeated) with concentration gradient of 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, reacted in boiling water bath immediately for 10min, cooled to room temperature (20 ℃), 200. mu.l of each reaction solution was added to a 96-well plate, and the light absorption value at 420nm wavelength was measured on a microplate reader, the results are shown in Table 2. By ddH₂O as blank control, with different concentrations of N-acetylglucosamine as abscissa, OD₄₂₀The absorbance values are plotted on the ordinate, and a standard curve is prepared, as shown in FIG. 2. Linearly fitting the N-acetylglucosamine standard curve to obtain a correlation coefficient R²0.9972, the linear regression equation is 0.6474-0.906 x.

TABLE 1 table for preparing series of concentration gradient standard solutions

	1	2	3	4	5	6	7
								10mMN-GlcNAc(mL)	0.01	0.02	0.03	0.04	0.05	0.06	0.07
ddH2O(mL)	0.99	0.98	0.97	0.96	0.95	0.94	0.93
								Schales’reagent(mL)	2	2	2	2	2	2	2

TABLE 2 relationship between reducing sugar content and light absorption value variation

	1	2	3	4	5	6	7
								N-GlcNAc(mM)	0.1	0.2	0.3	0.4	0.5	0.6	0.7
OD420	0.570	0.464	0.358	0.269	0.181	0.099	0.013

The enzymatic activity of the chitinase of example 2 was measured using 50g/l of colloidal chitin as a substrate. Taking 800 μ l substrate (pH5.0), adding 200 μ l chitinase crude enzyme solution, reacting in 45 deg.C water bath for 1h, and boiling in boiling water bath for 10min to inactivate enzyme. 1ml of Schales reagent was added and boiled at 100 ℃ for 5 min. Centrifuging at 12000rpm for 10min at 4 deg.C, taking enzyme solution with equivalent inactivation as blank control, and collecting supernatant to determine its absorbance at 420 nm. The reducing sugar content was calculated according to a standard curve (fig. 2). The specific activity of the enzyme was calculated to be 0.254U/ml.

The enzyme activity unit (U) is defined as: under the above conditions, the amount of enzyme required to release 1. mu. mol of reducing sugar per minute.

The product of enzymatic colloidal chitin was determined to be predominantly (GlcNAc)₂And (GlcNAc)₃With a small amount of GlcNAc, indicating that the chitinase of the invention is a typical endoplasmic endonuclease.

EXAMPLE 4 investigation of Endochostatin enzymatic Properties

The enzymatic properties of the chitinase of example 2 were measured, including optimum temperature, optimum pH, temperature stability, pH stability, influence of metal ions and chemical reagents on the enzymatic activity, and the enzymatic activity was measured using colloidal chitin (50g/l) as a substrate.

1. Optimum reaction temperature

The reaction system included 800. mu.l of colloidal chitin (50g/l) and 200. mu.l of crude enzyme solution of endochitinase. The pH was 5.0 (citric acid-sodium dihydrogen phosphate, 100 mM). Respectively reacting at 25 deg.C, 30 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 60 deg.C, 65 deg.C, 70 deg.C and 80 deg.C for 1h, taking the inactivated crude enzyme solution as blank, immediately adding 1m Schales reagent into the reaction system after reaction, boiling in boiling water bath for inactivation for 10min, and then placing in enzyme labeling instrument OD₅₄₀Then, the absorbance is measured, and the enzyme activity of the chitinase at different temperatures is determined. The optimum reaction temperature of the endoplasmic acid is determined by plotting the relative enzyme activity of the enzyme at different temperatures, as shown in FIG. 3. As can be seen from FIG. 3, the optimum reaction temperature for the chitinase was 45 ℃.

2. Temperature stability study

Respectively placing the crude chitin endonuclease solution at different temperatures (25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C), respectively incubating for 30min, 60min, 90min, and ice-cooling for 15min, detecting residual enzyme activity, comparing with untreated enzyme activity, and calculating relative enzyme activity. The results are shown in FIG. 4. The result shows that the chitinase has certain tolerance to temperature and good thermal stability at 20-50 ℃.

3. Optimum reaction pH

The enzyme activity is determined in a buffer solution with the pH value of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0(pH is 2.0-8.0 is citric acid-sodium dihydrogen phosphate buffer solution), 8.0, 9.0, 10, 11.0(pH is 8.0-11.0 is 100M of glycine-sodium hydroxide buffer solution). The reaction system is the same as that of the optimal reaction temperature test, the reaction temperature is 45 ℃, a curve is drawn according to the relative activity of the enzyme under different pH values, and the optimal reaction pH value is determined to be 5.0 as shown in figure 5.

4. Study of pH stability

The crude chitin enzyme solution is respectively placed in 100M buffer solutions with different pH values (pH 2.0-8.0 citric acid-sodium dihydrogen phosphate buffer solution, pH 8.0-12.0 glycine-sodium hydroxide buffer solution with an interval of 1.0) for 90min at 4 ℃, then the residual enzyme activity is detected at the optimal reaction temperature (45 ℃), and the relative enzyme activity is calculated by taking the untreated enzyme activity as a blank control, as shown in figure 6. The chitin incision enzyme has the highest enzyme activity at the pH value of 4.0-6.0, and has the best activity at the pH value of 5.0. The pH stability experiment shows that when the pH value of the chitinase is 4.0-6.0, the enzyme activity is maintained to be more than 90% after incubation for 90min, and the chitinase has higher pH tolerance.

5. Effect of Metal ions on chitinase Afchi18

CoCl with the final concentration of 0.01M is respectively added into the reaction systems (the same optimum reaction temperature test reaction system)₂、NaCl、MgCl₂、CuCl₂、FeCl₃、NH₄OH、CaCl₂、ZnCl₂、MnCl₂、KCl、BaCl₂Tris, SDS, EDTA, urea, and then enzyme activity was measured under standard conditions (45 ℃, pH5.0), and enzyme relative enzyme activity was calculated using untreated chitinase crude enzyme solution as a blank control, and the results are shown in fig. 7. The effect of metal ions on chitinase was calculated with the enzyme activity of the crude enzyme solution of untreated chitinase as 100%, and the results are shown in FIG. 7. As can be seen from FIG. 7, Co²⁺、Na⁺、Mg²⁺、NH₄ ⁺、Ca²⁺、Zn²⁺、K⁺Tris and urea have activation effect on chitinase; cu²⁺、Fe³⁺、Mn²⁺、Ba²⁺SDS and EDTA have inhibitory effect on chitinase.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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