Application of chitin exonuclease CmChi18A in degradation of chitin and chitin exonuclease mutant

Abstract

The invention relates to the technical field of gene engineering, in particular to application of chitin exonuclease CmChi18A in degradation of chitin and a chitin exonuclease mutant. The final product of the chitin subjected to enzymolysis by the chitin excision enzyme CmChi18A provided by the invention is N-acetyl-D-glucosamine, and the N-acetyl-D-glucosamine has the activity of chitobiose excision enzyme and the activity of beta-N-acetyl-D-glucosaminidase at the same time. According to the present invention, chitin exonuclease CmChi18A is subjected to directed evolution to prepare the forward mutant CmChi18Adx2 with the exonuclease activity as high as 12.33 U / mg, and the amino acid sequence of the mutant has no change compared with the chitin exonuclease CmChi18A, such that the basis is provided for efficient chitin degradation and mass N-acetyl-D-glucosamine preparation.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of chitin exonuclease CmChi18A in degrading chitin and a chitin exonuclease mutant. Background Art

[0002] Chitin, also known as chitosan, is composed of N-acetyl-D-glucosamine connected by β-1,4-glycosidic bonds. The biosynthesis of chitin in nature can reach 1×10 14 Tons, it is a polysaccharide second only to cellulose in nature, and is a huge and valuable biological resource. Chitin is widely distributed across multiple biological domains. Its main natural sources include the hard shells of crustaceans (such as shrimps, crabs, etc.); specific tissues in mollusks, such as squid cartilage; and cell walls in the fungal kingdom.

[0003] N-acetyl-D-glucosamine (GlcNAc), as a key product of chitin hydrolysis, has many important functions in the body. It plays a certain regulatory role in the immune system; it can be used as a dietary supplement, especially in the field of joint health; it has antioxidant and anti-inflammatory effects, can effectively reduce the production of free radicals and inflammatory reactions, and plays an important role in protecting cells and tissues from damage; it also has an inhibitory effect on the excessive growth of cancer cells or fibroblasts. These characteristics make them have a wide range of application potentials in many fields such as food science, medical health and cosmetics.

[0004] At present, the extraction technology for GlcNAc mainly covers three categories: chemical method, physical method and enzymatic method. Among them, chemical method, especially acid hydrolysis and oxidation method, occupies a dominant position in the preparation, but it cannot be ignored that these methods are often accompanied by high environmental pollution risks, safety hazards in the production process and relatively low yield, which limits its sustainable development potential. Physical methods, such as microwave treatment, ultrasonic assistance and electromagnetic wave radiation, have attracted attention for their simple operation and strong process controllability. However, physical methods are prone to produce more by-products during the treatment process, which affects the purity of the target product and the subsequent application effect. In contrast, enzymatic method is gradually becoming a hot spot in the research field with its significant advantages such as mild reaction conditions, no by-product generation and environmental friendliness. Enzymatic method can not only effectively retain the biological activity of GlcNAc, but also greatly reduce the environmental burden in the production process, opening up a new way for the green and efficient preparation of chitin hydrolysate. Therefore, in the future, enzymatic method is expected to occupy a more core position in the extraction and application of GlcNAc, and promote the green transformation and upgrading of related industries.

[0005] Chitinase (EC 3.2.1.14) is a glycoside hydrolase that can hydrolyze chitin. It can act on the β-1,4 glycosidic bond of chitin. Chitinase can be divided into endo-chitinase and exo-chitinase according to the different hydrolysis positions. Endochitinase shows a high degree of randomness and can randomly hydrolyze any glycosidic bond in the long chain of chitin, causing the long chain to break into shorter chitin oligosaccharides. In contrast, exo-chitinase is further refined into chitobiose exo-chitobiose and N-acetylglucosaminidase. The former can gradually release chitobiose from the reducing end or non-reducing end of chitin; the latter further cuts the chitobiose oligosaccharide to eventually generate GlcNAc monomers. If chitinase has both functions, the target product GlcNAc can be obtained by hydrolyzing the chitin substrate with a single enzyme. Not only is the condition mild and environmentally friendly, but the raw material cost is low and the output is efficient, which fully meets the core concepts and requirements of modern green industrial production.

[0006] Based on this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an application of chitin exonuclease CmChi18A in degrading chitin and a chitin exonuclease mutant.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides application of chitin exonuclease CmChi18A shown in SEQ ID No. 1 in degrading chitin to prepare N-acetyl-D-glucosamine.

[0010] Preferably, the chitin exonuclease CmChi18A degrades chitin by mixing the chitin exonuclease CmChi18A with chitin and reacting for 30 to 90 minutes.

[0011] Preferably, the chitin exonuclease CmChi18A and chitin are mixed in a mass ratio of 1:3 to 5;

[0012] The reaction temperature is 20-70°C;

[0013] The pH of the reaction is 3.0 to 8.0;

[0014] The reaction system also includes adding metal ions during the reaction;

[0015] The metal ions include NH4 + , Ca 2+ 、Zn 2+ , SDS, EDTA, Mg 2+ , Ba 2+ , Mn2+ and Li + One or more of .

[0016] Preferably, the nucleotide sequence encoding the chitin exonuclease CmChi18A shown in SEQ ID No.1 is shown in SEQ ID No.2.

[0017] Preferably, the primer pair for amplifying the nucleotide sequence shown in SEQ ID No. 2 is shown in SEQ ID Nos. 3 to 4.

[0018] Preferably, the reaction system for amplifying the nucleotide shown in SEQ ID No.2 is:

[0019] Template DNA 0.8-1.2 μL, upstream primer 0.8-1.2 μL, downstream primer 0.8-1.2 μL, 2× TaqMasterMix 24-26 μL, ddH2O2 0.4-23.6 μL;

[0020] The reaction conditions for amplifying the nucleotides shown in SEQ ID No.2 are:

[0021] Pre-denaturation at 95°C for 4 to 6 min; denaturation at 95°C for 29 to 31 s, annealing at 70°C for 29 to 31 s, extension at 72°C for 1.8 to 2.2 min, for a total of 35 cycles; post-extension at 72°C for 9 to 11 min.

[0022] The present invention also provides the use of the chitin exonuclease CmChi18A shown in SEQ ID No. 1 in preparing a product capable of degrading chitin.

[0023] The present invention also provides a mutant CmChi18A_dx2 of a chitin exonuclease CmChi18A, wherein the mutant CmChi18A_dx2 of the chitin exonuclease CmChi18A is obtained by directed evolution of the chitin exonuclease CmChi18A;

[0024] The amino acid sequence of the chitin exonuclease CmChi18A is shown in SEQ ID No.1;

[0025] The nucleotide sequence encoding the chitin exonuclease CmChi18A is shown in SEQ ID No.2;

[0026] The nucleotide sequence encoding the CmChi18A_dx2 is shown in SEQ ID No.5.

[0027] The present invention also provides a method for constructing a mutant of chitin exonuclease CmChi18A, comprising the following steps:

[0028] Using the nucleotide sequence encoding the chitin exonuclease CmChi18A as a template, error-prone PCR amplification is performed to collect forward mutants with improved chitin exonuclease activity;

[0029] The forward mutant with the greatest improvement in chitin exonuclease activity was CmChi18A_dx2;

[0030] The nucleotide sequence of the chitin exonuclease CmChi18A is shown in SEQ ID No.2;

[0031] The primer pairs used in the error-prone PCR amplification are shown in SEQ ID No. 6-7;

[0032] The reaction system of the error-prone PCR amplification is as follows: template DNA 0.8-1.2 μL; upstream primer 0.5 μL; downstream primer 0.5 μL; 50% glycerol 3.5-4.5 μL; 10× Taq Buffer (Mg 2+ free)1.8~2.2μL; Taq DNApolymerase1.0~1.4μL; Mg 2+ (50mM) 1.8~2.2μL; dNTP (2.5mM) 1.3~1.7μL; ddH2O 5.8~10.6μL;

[0033] The reaction conditions of the error-prone PCR amplification are: pre-denaturation at 95°C for 4.5-5.5 min; denaturation at 95°C for 28-32 s, annealing at 70°C for 28-32 s; extension at 72°C for 1.8-2.2 min, for a total of 35 cycles; and post-extension at 72°C for 9-11 min.

[0034] The present invention also provides the use of a mutant CmChi18A_dx2 of the chitin exonuclease CmChi18A or a forward mutant constructed according to the construction method in efficiently degrading chitin.

[0035] The present invention provides an application of chitin exonuclease CmChi18A in degrading chitin and a chitin exonuclease mutant. The final product of chitin hydrolysis by the chitin exonuclease CmChi18A provided by the present invention is N-acetyl-D-glucosamine, and the chitin exonuclease CmChi18A has chitobiose exonuclease activity and β-N-acetyl-D-glucosaminidase activity. The chitin exonuclease CmChi18A is subjected to directed evolution to prepare a forward mutant CmChi18A_dx2 with an exonuclease activity as high as 12.33U / mg. The amino acid sequence of the mutant is unchanged compared to the chitin exonuclease CmChi18A, providing a basis for efficiently degrading chitin and preparing N-acetyl-D-glucosamine in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The results of SDS-PAGE detection of chitin exonuclease CmChi18A. In the figure, M: marker protein, 1: total protein after cell lysis before centrifugation, 2: crude enzyme solution of chitin exonuclease CmChi18A, 3: protein collected during the loading process of total protein after cell lysis on Ni-NTABeads6FFcolumn, 4: impurity protein at the maximum UV reading after total protein after cell lysis was eluted with 100mL of 20mM imidazole, 5: impurity protein after 4, total protein after elution with 100mL of 20mM imidazole, 6: purified protein after 5, total protein after elution with 50mL of 400mM imidazole;

[0037] Figure 2 is the standard curve of N-acetyl-D-glucosamine;

[0038] Figure 3 This is the optimum temperature curve for the activity of chitin exonuclease CmChi18A;

[0039] Figure 4 This is the temperature stability curve of the activity of chitin exonuclease CmChi18A;

[0040] Figure 5 This is the optimum pH curve for the activity of chitin exonuclease CmChi18A;

[0041] Figure 6 This is the pH stability curve of the enzymatic activity of chitin exonuclease CmChi18A;

[0042] Figure 7 The effects of metal ions and chemical reagents on the activity of chitin exonuclease CmChi18A;

[0043] Figure 8 The effect of organic reagents on the activity of chitin exonuclease CmChi18A;

[0044] Fig. 9 Analysis of the hydrolysis products of chitin exonuclease CmChi18A;

[0045] Fig.10 The results of SDS-PAGE detection of chitin exonuclease CmChi18A and chitin exonuclease CmChi18A_dx2. In the figure, M: marker protein, 1: chitin exonuclease CmChi18A, 2: chitin exonuclease CmChi18A_dx2. DETAILED DESCRIPTION

[0046] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The amino acid sequences of chitin exonuclease CmChi18A and its mutant CmChi18A_dx2 are shown in SEQ ID No. 1. SEQ ID No. 1:.

[0048] Example 1

[0049] Construction of recombinant strain pET-28a(+)-CmChi18A / DH5α

[0050] Step 1: Design PCR primers

[0051] CmChi18A-F-5'-GTGCCGCGCGGCAGCCATATGACCGCTTGGAACAGCACCA-3' (SEQ ID No. 3);

[0052] CmChi18A-R-5'-GTGGTGGTGGTGGTGCTCGAGCTTGTTGATGTGAGTCATTGCTTCA-3' (SEQ ID No. 4).

[0053] Step 2: Amplify the CmChi18A gene by PCR using the primers in step 1

[0054] The primers in step 1 were used to amplify the gene of CmChi18A by PCR with (GenBank: CP157355) as the template. The reaction system for PCR amplification was: template 1 μL; upstream primer (SEQ ID No.3) 1 μL; downstream primer (SEQ ID No.4) 1 μL; 2×TaqMasterMix 25 μL; ddH2O2 2 μL; total reaction system volume 50 μL.

[0055] The reaction conditions of PCR amplification were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 70°C for 30 s; extension at 72°C for 2 min, for a total of 35 cycles; post-extension at 72°C for 10 min, and finally insulation at 16°C.

[0056] Step 3, after the PCR amplification is completed, the amplified product is recovered and purified to obtain the purified DNA product SEQ ID No.2.

[0057] Step 4, using restriction endonucleases NdeⅠ and XhoⅢ to digest the vector pET-28a(+), recovering the product, and obtaining the digestion-purified vector pET-28a(+) (5291 bp) (SEQ ID No.6).

[0058] Step 5, the DNA product SEQ ID No. 2 obtained in step 3 and the product purified by double enzyme digestion in step 4 are used Mix ligase was used for homologous recombination ligation.

[0059] The PCR homologous recombination ligation reaction system is: 1 μL of purified DNA product; 3 μL of vector pET-28a(+) after enzyme digestion and purification; Mix 5μL; ddH2O 1μL; total reaction system volume 10μL.

[0060] The reaction procedure of PCR homologous recombination connection is as follows: after the homologous recombination connection reaction system is mixed, it is placed in a 50°C water bath for 5 minutes, and then immediately placed on ice (0°C) for cooling, chemically transformed into E. coli DH5α, screened and sequenced by colony PCR, and a single colony is picked in LB medium and cultured overnight at 37°C and 220rpm to obtain the recombinant clone strain pET-28a(+)-CmChi18A / DH5α. The recombinant plasmid in the recombinant clone strain pET-28a(+)-CmChi18A / DH5α is extracted by StarPrepPlasmidMiniprepKit (GenStar), and the product obtained is the recombinant plasmid pET-28a(+)-CmChi18A.

[0061]

[0062]

[0063] Example 2

[0064] Expression of chitin exonuclease CmChi18A

[0065] Step 1, construct the recombinant expression strain pET28a-CmChi18A / E.coliBL21(DE3)

[0066] The recombinant plasmid pET-28a(+)-CmChi18A extracted in Example 1 was chemically transformed into the expression strain E. coli BL21-(DE3), and a single colony was picked and placed in LB medium, and cultured overnight at 37°C and 220rpm to obtain the recombinant expression strain pET28a-CmChi18A / E. coli BL21(DE3).

[0067] Step 2, expression of recombinant expression strain

[0068] The recombinant expression strain pET28a-CmChi18A / E.coliBL21(DE3) was inoculated into 10 mL of LB medium containing kanamycin sulfate (50 mg / mL), cultured overnight at 37°C for 16 h, and then inoculated into 1000 mL of LB medium containing kanamycin sulfate (50 mg / mL) at a 1% (v / v) inoculum, and cultured at 37°C until OD 600 =0.6, add isopropyl-β-D-thiogalactoside (IPTG) to make the final concentration in LB medium be 0.001M, and induce at 16°C, 200rpm for 24h.

[0069] Step 3, collecting chitinase crude enzyme solution

[0070] After the induction, the induced cultured bacterial liquid was centrifuged at 4°C, 8000rpm for 10 min to collect the bacteria, resuspended with citric acid-disodium hydrogen phosphate buffer, and ultrasonically disrupted on ice for 2s each time, with an interval of 2s, and ultrasonically disrupted for 20min; then, the supernatant was collected by centrifugation at 4°C, 12000rpm for 30min to obtain the crude enzyme solution of chitin exonuclease CmChi18A.

[0071] Step 4, purification of crude chitin enzyme solution

[0072] The crude enzyme solution of chitin exonuclease CmChi18A in step 4 was purified using a Ni-NTABeads6FFcolumn column and eluted with imidazole gradient. The purified protein after elution with 400 mM imidazole was collected as chitin exonuclease CmChi18A.

[0073] The protein solutions at different stages were collected for SDS-PAGE. Figure 1 shown.

[0074] Figure 1 It was shown that the size of the chitin exonuclease CmChi18A after elution with 400 mM imidazole was 77.39 kDa. After sequence determination, the amino acid sequence of the chitin exonuclease CmChi18A was shown in SEQ ID No. 1, and SEQ ID No. 1 was:.

[0075] Example 3

[0076] Enzymatic properties of chitin exonuclease CmChi18A

[0077] (1) Preparation of standard curve using 3,5-dinitrosalicylic acid (DNS) method

[0078] Drawing of the standard curve of N-acetyl-D-glucosamine: accurately weigh a certain amount of N-acetyl-D-glucosamine (GlcNAc), dissolve it in ddH2O, and prepare a mother solution with a concentration of 100mM; continue to dilute to a final concentration of 10mM GlcNAc standard reagent for use. According to Table 1, add N-acetyl-D-glucosamine standard reagent (10mM GlcNAc) and ddH2O in sequence, mix well, and obtain a series of concentration gradients of 0.4mM, 0.6mM, 0.8mM, 1mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM, 2mM, 2.2mM, 2.4mM, 2.6mM, 2.8mM, 3mM, 3.2mM, 3.4mM GlcNAc standard solutions (3 replicates per group), add 0.5mL DNS reagent to each experimental group, immediately react in a boiling water bath for 5min, and cool to room temperature in an ice bath. 200 μL of the reaction solution was added to a 96-well ELISA plate in each experimental group, and the absorbance was read at a wavelength of 540 nm on an ELISA reader. The results are shown in Table 2. ddH2O was used as a blank control, and different concentrations of N-acetyl-D-glucosamine were used as the horizontal axis. OD 540 The absorbance value is the ordinate, and a standard curve is made, such as Figure 2 The linear fitting of the N-acetyl-D-glucosamine standard curve gave a correlation coefficient of R 2 =0.9990, the linear regression equation is y=0.6409x-0.1818.

[0079] Table 1 Series concentration gradient standard solution configuration table

[0080] 1 2 3 4 5 6 7 8 10mM N-GlcNAc (μL) 20 30 40 50 60 70 80 90 <![CDATA[ddH2O(μL)]]> 480 470 460 450 440 430 420 410 DNS(μL) 500 500 500 500 500 500 500 500 9 10 11 12 13 14 15 16 10mM N-GlcNAc (μL) 100 110 120 130 140 150 160 170 <![CDATA[ddH2O(μL)]]> 400 390 380 370 360 350 340 330 DNS(μL) 500 500 500 500 500 500 500 500

[0081] Table 2 Corresponding relationship between reducing sugar content and absorbance value change

[0082] 1 2 3 4 5 6 7 8 N-GlcNAc(mM) 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 <![CDATA[OD 540 ]]> 0.093 0.198 0.318 0.445 0.601 0.711 0.829 0.966 9 10 11 12 13 14 15 16 N-GlcNAc(mM) 2 2.2 2.4 2.6 2.8 3 3.2 3.4 <![CDATA[OD 540 ]]> 1.08 1.233 1.367 1.502 1.628 1.784 1.886 1.965

[0083] (2) Enzyme activity determination

[0084] Using 1% colloidal chitin as substrate, the enzyme activity of the chitin exonuclease CmChi18A prepared in Example 2 was determined. Take 400 μL of substrate (pH 6.5), add 100 μL of chitin exonuclease solution prepared in Example 2, react in a 50°C water bath for 10 minutes, then move to a boiling water bath and boil for 10 minutes to inactivate the enzyme. Add 0.5 mL of DNS reagent and boil at 100°C for 5 minutes. Centrifuge at 4°C, 12000 rpm for 10 minutes in a centrifuge. Use an equal amount of citric acid-disodium hydrogen phosphate (100 mM, pH 6.5) as a blank control, take the supernatant and determine its absorbance at 540 nm. According to the standard curve ( Figure 2 ) Calculate the reducing sugar content. The activity of chitin exonuclease CmChi18A was calculated to be 9.52 U / mg.

[0085] The enzyme activity unit (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the above conditions.

[0086] It was determined that the products of chitin exoenzyme CmChi18A hydrolyzing colloidal chitin were mainly GlcNAc and (GlcNAc)2, and the final product was GlcNAc, indicating that the chitinase of the present invention has the properties of a typical chitobiose exoenzyme and has the properties of β-N-acetyl-D-glucosaminidase.

[0087] Example 4

[0088] The enzymatic properties of the chitin exonuclease CmChi18A prepared in Example 2 were measured, including the optimum temperature, optimum pH, temperature stability, pH stability, the effects of metal ions, chemical reagents, and organic reagents on enzyme activity, hydrolysis product analysis, and enzyme activity was measured using colloidal chitin (1%) as a substrate.

[0089] 1. Optimum reaction temperature

[0090] The reaction system includes 400 μL colloidal chitin (1%) and 100 μL chitin exonuclease CmChi18A. The pH value is 6.5 (citric acid-disodium hydrogen phosphate, 100 mM). The reaction was carried out at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 10 min, and an equal amount of citric acid-disodium hydrogen phosphate (100 mM, pH 6.5) buffer was added as a blank control. After the reaction, 0.5 mL DNS reagent was immediately added to the reaction system, boiled in a boiling water bath for 5 min, and immediately cooled to room temperature. After centrifugation, the supernatant was taken and measured on an OD reader. 540 The absorbance was measured at different temperatures to determine the enzymatic activity of chitin exonuclease at different temperatures. A curve was drawn based on the relative enzymatic activity of the enzyme at different temperatures, such as Figure 3 The optimal reaction temperature of chitin exonuclease CmChi18A is 50℃.

[0091] 2. Temperature stability study

[0092] Chitin exonuclease CmChi18A was incubated at different temperatures (20°C, 30°C, 40°C, 45°C, 50°C, 60°C, 70°C) for 30 min and 90 min, respectively, and then the residual enzyme activity was detected. Figure 4 As shown, this indicates that chitin exonuclease CmChi18A has a certain tolerance to temperature and has good thermal stability within 20-40°C.

[0093] 3. Optimal reaction pH

[0094] The substrate is 1% colloidal chitin, and the enzymatic activity of chitin exonuclease CmChi18A is measured in buffer solutions with pH values ​​of 3.0, 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0 (pH 3.0-8.0 is 100mM citric acid-sodium dihydrogen phosphate buffer solution), 8.0, 9.0, 10.0, 11.0, 12.0 (pH 8.0-12.0 is 100mM glycine-sodium hydroxide buffer solution). The reaction system is the same as that of the optimal reaction temperature test, and the reaction temperature is 50°C. The relative activity curve of the enzyme at different pH values ​​is drawn, such as Figure 5 As shown, the optimal reaction pH value of chitin exonuclease CmChi18A was determined to be 6.5.

[0095] 4. pH stability study

[0096] Chitin exonuclease CmChi18A was placed in 100 mM buffer solutions of different pH values ​​(pH 3.0-8.0 citric acid-sodium hydrogen phosphate buffer solution, pH 8.0-12.0 glycine-sodium hydroxide buffer solution) at 4°C for 180 min, and then the residual enzyme activity was detected at the optimal reaction temperature (50°C). Figure 6 As shown, the chitin exonuclease CmChi18A has the highest enzyme activity between pH 6.0 and 7.5, and the best activity at pH 6.0. The pH stability experiment shows that the chitin exonuclease CmChi18A has a high pH tolerance when the pH value ranges from 6.0 to 7.5, and the enzyme activity is maintained above 80% after incubation for 180 minutes.

[0097] 5. Effects of metal ions on chitin exonuclease

[0098] FeCl3, NH4Cl, NaCl, CuCl2, NiCl2, CoCl2, ZnCl2, urea, MgCl2, BaCl2, MnCl2, CmCl2, KCl, SDS, EDTA, LiCl were added to the reaction system (same as the reaction system of the optimal reaction temperature test) with a final concentration of 10mM, and then the enzyme activity was determined under standard conditions (50℃, pH6.5). The relative enzyme activity was calculated with the untreated chitinase activity as 100% (control). The results are shown in Figure 7 As shown, NH4 + , Ca 2+ 、Zn 2+ , SDS, EDTA, Mg 2+ , Ba 2+ , Mn 2+ , Li + It has an activating effect on the chitin exonuclease CmChi18A, Fe 3+ , Cu2+ It has an inhibitory effect on chitin exonuclease CmChi18A.

[0099] 6. Effects of organic reagents on chitin exonuclease

[0100] To the reaction system (same as the optimal reaction temperature experiment reaction system), 1% (v / v) of glycerol, propylene glycol, n-butanol, ethyl acetate, isopropanol, methanol, and acetonitrile were added respectively, and then the residual enzyme activity was measured under standard conditions (50°C, pH 6.5), and the relative enzyme activity was calculated with the untreated chitinase activity as 100% (control). The results are shown in Figure 8 As shown. Figure 8 It can be seen that the chitin exonuclease CmChi18A has an activating effect, while chloroform, ethyl acetate, ethanol, formic acid, glycerol, methanol and acetonitrile have an inhibitory effect on the chitin exonuclease CmChi18A.

[0101] From the above examples, it can be seen that the size of chitin exonuclease CmChi18A is 77.39 kDa, the optimal reaction temperature of the enzyme is 50°C, and the thermal stability is good in the range of 20°C to 40°C; the optimal pH is 6.5, and the enzyme activity is high at pH 5.0 to 6.5, indicating that the chitinase provided by the present invention has high stability. The metal ion and chemical reagent effect test showed that Fe 3+ NH4 + 、Na + , Cu 2+ 、Ni 2+ 、Co 2+ 、Zn 2+ 、Urea、Mg 2+ , Ba 2+ , Mn 2+ , Ca 2+ , Li + It has an activating effect on the chitin exonuclease CmChi18A, K + , SDS, and EDTA have an inhibitory effect on chitin exonuclease CmChi18A. The effect experiment of organic reagents showed that organic reagents had no activating effect on exonuclease CmChi18A, ethyl acetate and n-butanol had an inhibitory effect on chitin exonuclease, and glycerol, acetonitrile and 1,2-propylene glycol had little effect on chitin exonuclease CmChi18A.

[0102] 7. Analysis of hydrolysis products

[0103] 1 mL of the chitin exonuclease CmChi18A solution prepared in Example 2 with a concentration of 1 mg / mL was mixed with 3 mL of 1% colloidal chitin and reacted at 30°C for different time periods (10 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h), samples were taken at different time intervals and boiled for 10 min to inactivate, centrifuged at 12000 rpm for 5 min, and the supernatant was the enzyme reaction product. The enzyme reaction product was used with The analysis was performed using an HPLC system (UltiMate 3000 series) with a 5 μm NH2 column (LC Column 250×4.6 mm) and a differential refractive index detector (RID). The mobile phase was 70% acetonitrile at a flow rate of 0.5 mL / min.

[0104] Specific results such as Fig. 9 shown.

[0105] Fig. 9 It shows that in the first 2 hours of the reaction, the main product of chitinase CmChi18A hydrolyzing colloidal chitin is (ClcNAc)2, and then the GlcNAc content increases. When the reaction is carried out for 24 hours, the main product is GlcNAc, indicating that chitinase CmChi18A has both the activity of chitobiose exonuclease and the activity of β-N-acetyl-D-glucosaminidase.

[0106] Example 5

[0107] Directed evolution of chitin exonuclease CmChi18A

[0108] CmChi18A was subjected to directed evolution. The chitinase CmChi18A gene (SEQ ID No. 2) purified in Example 2 was used as a template, and primers CmChi18A-F (SEQ ID No. 3) and CmChi18A-R (SEQ ID No. 4) were used for error-prone PCR. The reaction system for error-prone PCR amplification was: template 1 μL; upstream primer (SEQ ID No. 3) 0.5 μL; downstream primer (SEQ ID No. 4) 0.5 μL; 50% glycerol 4 μL; 10× TaqBuffer (Mg 2+ free)2μL; TaqDNApolymerase1.2μL; Mg 2 + (50mM) 2μL; dNTP (2.5mM) 1.5μL; ddH2O 8.3μL; total reaction volume 20μL. The reaction conditions for PCR amplification were: 95℃ pre-denaturation for 5min; 95℃ denaturation for 30s, 70℃ annealing for 30s; 72℃ extension for 2min, a total of 35 cycles; 72℃ post-extension for 10min, and finally 16℃ incubation.

[0109] The subsequent steps are the same as steps 3 to 5 of Example 1 and Example 2.

[0110] Through enzyme activity verification, a total of 5 positive mutants with improved enzyme activity were screened and shown in Table 3. Among them, the mutant with the highest enzyme activity was named CmChi18A_dx2. After enzyme activity determination, the enzyme activity of the mutant increased by 30%, the enzyme activity of CmChi18A was 9.58U / mg, and the enzyme activity of the mutant CmChi18A_dx2 was increased to 12.33U / mg. The amino acid sequence of CmChi18A_dx2 did not change through sequencing analysis (as shown in SEQ ID No.1), but the nucleotide sequence changed as shown in SEQ ID No.5. In order to further verify the purity difference between the wild enzyme and its mutant, SDS-PAGE technology was used for analysis, and the results are shown in Fig.10 In. Fig.10 As shown in the electrophoresis bands of CmChi18A (lane 1) and CmChi18A_dx2 (lane 2), it can be clearly observed that the band of CmChi18A_dx2 is more single and has a deeper concentration, which indicates that the forward mutant exhibits a higher purity than the wild-type enzyme. This shows that the main reason for the increase in the specific activity of this forward mutant is that a higher purity recombinant enzyme CmChi18A_dx2 can be obtained. Therefore, the high purity characteristics of CmChi18A_dx2 indicate that it may have a broader application prospect in the fields of enzyme catalysis and high-value utilization of chitin.

[0111]

[0112] Table 3 Enzyme activities of directed evolution mutants

[0113] serial number Enzyme activity (U / mg) CmChi18A 9.58 CmChi18A_dx2 12.33 CmChi18A_dx7 9.72 CmChi18A_dx16 11.09 CmChi18A_dx33 10.21 CmChi18A_dx35 9.69

[0114] As can be seen from the above embodiments, the present invention provides an application of chitin exonuclease CmChi18A in the degradation of chitin and a chitin exonuclease mutant. The final product of chitin hydrolysis by the chitin exonuclease CmChi18A provided by the present invention is N-acetyl-D-glucosamine, and it has chitobiose exonuclease activity and β-N-acetyl-D-glucosaminidase activity. The chitin exonuclease CmChi18A is subjected to directed evolution to prepare a forward mutant CmChi18A_dx2 with an exonuclease activity as high as 12.33U / mg. The amino acid sequence of the mutant is unchanged compared to the chitin exonuclease CmChi18A, providing a basis for efficiently degrading chitin and preparing N-acetyl-D-glucosamine in large quantities.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of chitin exonuclease CmChi18A shown in SEQ ID No.1 in the preparation of N-acetyl-D-glucosamine by degrading chitin.

2. The use according to claim 1, characterized in that: The method of chitin exonuclease CmChi18A for degrading chitin is as follows: chitin exonuclease CmChi18A is mixed with chitin and reacted for 10 to 90 minutes.

3. The use according to claim 2, characterized in that: The chitin exonuclease CmChi18A and chitin are mixed in a mass ratio of 1:3-5; The reaction temperature is 20-70°C; The pH of the reaction is 3.0 to 8.0; During the reaction, the reaction system also includes adding metal ions; The metal ions include NH4 + , Ca 2+ 、Zn 2+ , SDS, EDTA, Mg 2+ , Ba 2+ , Mn 2+ and Li + One or more of .

4. The use according to claim 3, characterized in that: The nucleotide sequence encoding the chitin exonuclease CmChi18A shown in SEQ ID No.1 is shown in SEQ ID No.

2.

5. The use according to claim 4, characterized in that: The primer pair for amplifying the nucleotide sequence shown in SEQ ID No. 2 is shown in SEQ ID Nos. 3-4.

6. The use according to claim 5, characterized in that: The reaction system for amplifying the nucleotide shown in SEQ ID No.2 is: Template DNA 0.8-1.2 μL, upstream primer 0.8-1.2 μL, downstream primer 0.8-1.2 μL, 2×Taq MasterMix 24-26 μL, ddH2O 20.4-23.6 μL; The reaction conditions for amplifying the nucleotides shown in SEQ ID No.2 are: Pre-denaturation at 95°C for 4 to 6 min; denaturation at 95°C for 29 to 31 s, annealing at 70°C for 29 to 31 s, extension at 72°C for 1.8 to 2.2 min, for a total of 35 cycles; post-extension at 72°C for 9 to 11 min.

7. Application of the chitin exonuclease CmChi18A shown in SEQ ID No.1 in the preparation of products capable of degrading chitin.

8. A mutant CmChi18A_dx2 of chitin exonuclease CmChi18A, characterized in that: The chitin exonuclease CmChi18A mutant CmChi18A_dx2 is obtained by directed evolution of the chitin exonuclease CmChi18A; The amino acid sequence of the chitin exonuclease CmChi18A is shown in SEQ ID No.1; The nucleotide sequence encoding the chitin exonuclease CmChi18A is shown in SEQ ID No.2; The nucleotide sequence encoding the CmChi18A_dx2 is shown in SEQ ID No.

5.

9. A method for constructing a mutant of chitin exonuclease CmChi18A, characterized in that: The steps include: Using the nucleotide sequence encoding the chitin exonuclease CmChi18A as a template, error-prone PCR amplification is performed to collect forward mutants with improved chitin exonuclease activity; The forward mutant with the greatest improvement in chitin exonuclease activity was CmChi18A_dx2; The nucleotide sequence of the chitin exonuclease CmChi18A is shown in SEQ ID No.2; The primer pairs used in the error-prone PCR amplification are shown in SEQ ID No. 6-7; The reaction system of the error-prone PCR amplification is as follows: template DNA 0.8-1.2 μL; upstream primer 0.5 μL; downstream primer 0.5 μL; 50% glycerol 3.5-4.5 μL; 10× Taq Buffer (Mg 2+ free)1.8~2.2μL; Taq DNApolymerase 1.0~1.4μL; Mg 2+ (50mM) 1.8~2.2μL; dNTP (2.5mM) 1.3~1.7μL; ddH2O 5.8~10.6μL; The reaction conditions of the error-prone PCR amplification are: pre-denaturation at 95°C for 4.5-5.5 min; denaturation at 95°C for 28-32 s, annealing at 70°C for 28-32 s; extension at 72°C for 1.8-2.2 min, for a total of 35 cycles; and post-extension at 72°C for 9-11 min.

10. Use of the mutant CmChi18A_dx2 of the chitin exonuclease CmChi18A according to claim 8 or the forward mutant constructed according to the construction method according to claim 9 in efficiently degrading chitin.

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