Cloning and expression of an enzyme chil capable of efficiently degrading chitin powder and its application

Abstract

The application discloses a kind of enzyme Chi1 cloning expression and its application that can degrade powder particle chitin efficiently.The coding gene of the enzyme is derived from chitin-degrading bacteria Chitinibacter Sp.GC72, belongs to the chitinase of glycoside hydrolase 18 family, is heterologously expressed in Escherichia coli, and the obtained recombinant enzyme exhibits excellent activity at 30-40 DEG C and pH 4.0-10.0, and has good environmental tolerance.The chitinase Chi1 can efficiently degrade chitin substrates, and can also directly degrade biomass containing chitin such as lobster shell, Chinese mitten crab shell and ganoderma, and the product of degrading chitin is mainly (GlcNAc) 2, with a small amount of GlcNAc and (GlcNAc) 3.Among them, when degrading colloidal chitin and powder particle chitin, the enzyme activity reaches 493.73 U / mg and 91.76 U / mg respectively, which is the highest level at present.In addition, by using chitooligosaccharide as a substrate to study the hydrolysis mode of Chi1, it is confirmed that Chi1 has double functions, and exhibits exoactivity and N-acetylglucosaminidase activity.The application provides a new direction for efficient degradation of powder particle chitin and waste chitin-rich material, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis, specifically relating to the cloning, expression, and application of an enzyme Chi1 that can efficiently degrade chitin in powder particles. Background Technology

[0002] Chitin, also known as chitosan, is composed of... N - Acetaminophen (GlcNAc) via β A natural polymer formed by -D-(1,4)-glycosidic bonds, with the molecular formula (C8H2O) 13 05N) n With a relative molecular weight exceeding 1 million, chitin is second only to cellulose in terms of abundance on Earth. It is primarily found in the shells of crustaceans, the exoskeletons of insects, and the cell walls of fungi. However, due to the lack of efficient refining methods, it is typically disposed of as waste through landfills or dumping, not only taking up space but also imposing a severe burden on the environment. Therefore, the development and efficient utilization of chitin resources is crucial. However, chitin's insolubility in water is one of the biggest obstacles to its development and utilization. [The text then abruptly shifts to a seemingly unrelated topic:] ...to extract the... β Cleavage of the -1,4-glycosidic bond to form soluble GlcNAc and low-polymerization-degree chitin oligosaccharide products is one of the main strategies for achieving efficient utilization of chitin.

[0003] Chitosan oligosaccharides and GlcNAc possess excellent biological functions, including antibacterial, antitumor, immunomodulatory, and wound-healing properties. They have significant applications in agriculture, medicine, food, and feed additives. Therefore, converting discarded chitin resources into high-value-added chitosan oligosaccharides and GlcNAc is crucial for their utilization, bringing both economic and environmental benefits.

[0004] Currently, the conversion of chitin into chitosan oligosaccharides and GlcNAc mainly utilizes acid hydrolysis. However, the resulting products are of poor quality, prone to deacetylation, and generate large amounts of acidic and alkaline wastewater, causing serious environmental pollution. Enzymatic hydrolysis, on the other hand, is a green, low-energy-consumption, and highly specific method, making it the preferred approach for converting chitin into chitosan oligosaccharides and GlcNAc. It has become a hot topic in chitin hydrolysis research in recent years. Chitinases are widely found in bacteria, fungi, and plants and animals, but their activity is generally low, resulting in poor chitin hydrolysis efficiency. Natural chitin (α-configuration, mostly in powder form) has extremely high crystallinity, making enzymatic hydrolysis of chitin still challenging at present. Therefore, identifying chitinases that can efficiently degrade powdered chitin and its enrichments, and using them to prepare high-value-added chitosan oligosaccharides and GlcNAc, is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an enzyme Chi1 that can efficiently degrade chitin in powder particles. This gene is derived from a bacterial strain. Chitinibacter sp . GC72, after expression in E. coli, showed good thermostability of the recombinant enzyme Chi1 at 25-40℃; it also exhibited high activity at pH 4.0-10.0, indicating high stability of Chi1; Mg metal ion 2+ Ba 2+ Na + Ca 2+ and Li + It promotes enzyme activity; the specific enzyme activities for colloidal chitin and particulate chitin are 493.73 U / mg and 91.76 U / mg, respectively. It can also degrade chitin-rich biomass, such as lobster shells and crab shells. The main product of chitin hydrolysis by Chi1 is (GlcNAc)2, with small amounts of GlcNAc and (GlcNAc)3. Analysis of the chitin oligosaccharide hydrolysis pattern shows that Chi1 is a bifunctional enzyme, possessing both exonuclease and N-acetylglucosidase activities.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] An enzyme Chi1 that can efficiently degrade chitin in powder particles, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] SEQ ID No. 2:

[0009] ASFAATAWNSTTVYTGGEIVTYQGTDYKAKWWTQGNVPGAEQWGPWEAQGPVSATPVPTATTAPTATPVTTATSAPTATPKPTATPAVGTCVDAAWNSSTAYTGGQKVSYNGRTYQAQWWTSGDQPDLNTGSGKPWKDLGACGPVVATPTPTATVAPTATPVVTATPTPTATPVVTATPTATPVGPTPTPTPVVTPTVPPSTGAKQVGTYFAEWSIYGRKFFLKNVQDSGQAAKLTFLNYSFGNVYKQADGTYKCQANINKAETGNQDGGDAWALYQKGFAANESVDGVADAWGQDGKGSLKGNWNQLKKLKAKNPNMKVLISLGGWTWSKWFSAAASTDALRKTLVASCIDVWIKGNLPFDAASNAGGAGTGAGVFDGIDIDWEYPGVQGIGTNTVSPADKENNTLLMKEFREQLDAIGSQTGKRYLLTVAIGAGDEKIAATVPGEYSKYLDWINIMSYDYNGGWDAAGPTDFQSNLYQDPASPRTVDPKTGKVSKYYTDAAVKDLIARGVPAVKLHIGVPFYGRGWTGVTNVNNGLYQKATGAAKGTYESGIEDYKVLKTAPGTEYIHPVTQQTYKFDGSTFWSYDTPRDIKLKADYAKSMGMGGIFSWEADGDTANGELVEAMTHINK

[0010] As an improvement, the nucleotide sequence encoding the highly efficient degradable powder granule endochitinase Chi1 is as shown in SEQ ID NO: 1.

[0011] SEQ ID No.1:

[0012]

[0013] A recombinant plasmid containing a nucleotide sequence encoding Chi1, an enzyme capable of efficiently degrading chitin in powder particles.

[0014] A recombinant strain expressing the above-mentioned recombinant plasmid.

[0015] The method for constructing the above recombinant strain includes the following steps:

[0016] Step 1: Design primers and amplify the sequence shown in SEQ ID NO:1 by PCR;

[0017] Step 2, construct the recombinant plasmid pET-28a(+)- Chi1 ;

[0018] Step 3, the obtained recombinant plasmid pET-28a(+)- Chi1 Introducing the recombinant plasmid pET-28a(+) into E. coli BL21(DE3) yielded a result containing the recombinant plasmid pET-28a(+). -Chi1 Escherichia coli BL21(DE3);

[0019] Step 4: Select single clones of recombinant bacteria, culture them overnight on a shaker, add an inducer to induce culture, and collect the bacterial cells after centrifugation;

[0020] Step 5: Resuspend the bacteria in buffer solution, then sonicate to lyse, centrifuge to collect the supernatant and freeze for later use.

[0021] The application of the chitin endonuclease Chi1 in the degradation of colloidal chitin and powder chitin.

[0022] As an improvement, the enzymatic hydrolysis temperature in the application is 30-55℃ and the pH is 3.0-11.0, wherein the optimal temperature and optimal pH for enzymatic hydrolysis are 40℃ and 7.0, respectively, and the stability is good at 30-40℃ and pH 4.0-10.0.

[0023] As an improvement, Mg in the aforementioned application 2+ Ba 2+ Na + Ca 2+ and Li + The effect of promoting the activity of Chi1 during enzymatic hydrolysis.

[0024] As an improvement, when chitinase Chi1 degrades colloidal chitin and powdered chitin, the enzyme activity reaches 493.73 U / mg and 91.76 U / mg, respectively, and the hydrolysis product is mainly (GlcNAc)2, accompanied by a small amount of GlcNAc and (GlcNAc)3.

[0025] As an improvement, the concentration of colloidal chitin or powdered chitin is 5-100 g / L.

[0026] Beneficial effects:

[0027] Compared with existing technologies, the present invention provides a Chi1 enzyme cloning and expression method for efficiently degrading chitin in powder particles, and its application, which has the following advantages:

[0028] Heterologous expression in Escherichia coli resulted in high soluble expression and good activity.

[0029] 2. Chitinase Chi1 has excellent enzymatic properties, good environmental tolerance, and good prospects for industrial application.

[0030] 3. Chitinase Chi1 has a specific enzyme activity of 91.76 U / mg for highly crystalline natural powder chitin, which is the highest level to date. It can also degrade biomass containing chitin.

[0031] 4. Chi1 is a bifunctional chitinase with exonuclease and N-acetylglucosamine activities. It can simultaneously produce GlcNAc and chitobiose, providing a new direction for the efficient, green, and high-value utilization of natural powdered chitin and waste shrimp and crab shells, and has good development prospects. Attached Figure Description

[0032] Figure 1 The SDS-PAGE results of the chitinase Chi1, which can efficiently degrade particulate chitin according to the present invention, are shown. M is the protein standard molecular weight marker, 1 is the supernatant obtained by centrifuging Chi1 protein, and 2 is the purified Chi1 protein.

[0033] Figure 2 This invention illustrates the effect of temperature on the activity of chitinase Chi1; where (a) represents the optimum temperature and (b) represents temperature stability.

[0034] Figure 3 The effect of pH on the activity of chitinase Chi1 in this invention; where (a) is the optimal pH; and (b) is the pH stability.

[0035] Figure 4 The effect of metal ions on the activity of chitinase Chi1;

[0036] Figure 5 Analysis of the results of chitinase Chi1 degradation of chitin products in this invention;

[0037] Figure 6 This is an example of the degradation of chitin oligosaccharide products by the chitinase Chi1 in this invention, including (a) chitobiose, (b) chitotetraose, and (c) chitohexaose. Detailed Implementation

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art described herein. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0039] The present invention will be further described below through embodiments, but these are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0042] The colloidal chitin used in the following examples was prepared by the following method: 5 g of powdered chitin was added to...

[0043] Stir thoroughly in 60 mL of concentrated hydrochloric acid until a paste is formed. After standing for 1-2 hours, slowly add the solution to 2000 mL of 95% ethanol solution while stirring vigorously. Let stand overnight to obtain precipitated colloidal chitin. Rinse repeatedly with sterile water, adjust the pH to 7, and then make up to volume with buffer solution. Example 1

[0044] The strains used in this invention Chitinibacter sp . GC72 was screened and deposited by our laboratory at the China Center for Type Culture Collection (CCTCC), the China Center for Type Microbial Culture Collection, in 2014, with accession number NO.2014113.

[0045] The plasmids, Escherichia coli BL21 (DE3) and other materials used in this invention are all commercially available and conventional. Any technical means not mentioned are conventional in the field and will not be described in detail here. Example 2

[0046] Recombinant plasmid pET-28a(+)- Chi1 Construction

[0047] Step 1: Design PCR amplification primers

[0048] Chi1- F-5'- GAACAGATTGGTGCA GGATCCGTCAGCTTCTTTCGCTGCT-3';

[0049] Chi1- R-5'- GTGGTGGTGGTGGTG CTCGAG CTTGTTGATGTGAGTCAT-3',

[0050] The full length of Chi1 was amplified;

[0051] Step 2, the reaction conditions for the above PCR amplification are as follows:

[0052] Pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 s, annealing at 60 ℃ for 5 s, extension at 72 ℃ for 10 s, for 30 cycles; final extension at 72 ℃ for 10 min. The amplified product was recovered after amplification.

[0053] Step 3: Digest the vector pET-28a(+) with restriction endonucleases BamHI and XhoI, and then recover the vector.

[0054] Step 4: Homologous recombination ligation is performed between the amplification product obtained in Step 3 and the vector obtained in Step 4 to obtain the recombinant plasmid pET-28a(+)- Chi1 ;

[0055] Step 5, the recombinant plasmid pET-28a(+)- obtained in step 4 Chi1 Colony PCR was performed using universal primers, and the correct bands were sequenced. Example 3

[0056] Construction of recombinant expression strain pET-28a(+)- Chi1- E. coli BL21(DE3)

[0057] The first step is to recombinant plasmid pET-28a(+)- Chi1 Introducing Escherichia coli BL21(DE3) yielded a sample containing the recombinant plasmid pET-28a(+)- Chi1 Escherichia coli BL21(DE3);

[0058] The second step is to select recombinant bacteria pET-28a(+)- Chi1 The single clones were inoculated into 5 mL LB medium containing 100 μg / mL kanamycin and cultured overnight in a shaking incubator at 37°C and 200 rpm.

[0059] The third step involves inoculating the above bacterial suspension into 100 mL of LB medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, and incubating at 37°C with shaking at 200 rpm until OD (out of control) is reached. 600Reaching 0.6-0.8;

[0060] Fourth step, add IPTG (inducer) to a final concentration of 1 mmol / L, and incubate at 18°C ​​and 200 rpm with shaking for 20 h;

[0061] Step 5: Collect the above bacterial culture into a centrifuge tube, centrifuge at 4°C and 6000 rpm for 10 min, discard the supernatant and collect the precipitate;

[0062] Step 6: Add 10 mL of PBS buffer (pH 7.0, 50 mM) to the precipitate to resuspend the bacterial cells;

[0063] Step 7: Bacterial lysis by ultrasound on ice. The ultrasound power is 300 W, each ultrasound treatment lasts 2 seconds, with a 3-second interval, for a total of 10 minutes of ultrasound treatment.

[0064] Step 8: Centrifuge at 8000 rpm for 15 min at 4℃, collect the supernatant of the lysis buffer, and place it on ice;

[0065] Step 9: The collected supernatant was purified using nickel ion affinity chromatography via an AKTA protein purification system. The purification process was performed on an AKTA protein purifier. The binding buffer contained 25 mmol / L imidazole, and the elution buffer contained 250 mmol / L imidazole. The purified recombinant protein solution was then subjected to SDS-PAGE, yielding a protein band of approximately 95 kDa (results are shown below). Figure 1 (as shown) Figure 1 From left to right in the middle are: Marker, crude enzyme solution obtained from the supernatant in step 8 of Example 3, and pure enzyme obtained from step 9 of Example 3. Example 4

[0066] Enzyme activity assay of Chi1, an enzyme that can efficiently degrade powder particles.

[0067] 1. DNS reagent preparation

[0068] Weigh 182.0 g of potassium sodium tartrate, add 0.5 L of pure water, heat to dissolve at 50 ℃, then add 6.3 g of 3,5-dinitrosalicylic acid (DNS), 21.0 g of sodium hydroxide, and 5.0 g of phenol, stir until completely dissolved, cool, add pure water to make up to 1.0 L, store in the dark, and let stand for 7 days before use.

[0069] 2. Establishment of the standard curve for enzyme activity assay

[0070] Chi1 enzyme activity was determined using the DNS (3,5-dinitrosalicylic acid) method. 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of N-acetylglucosamine standard solution (1 mg / mL) were respectively placed in 4 mL tubes, and diluted to 1.0 mL with pure water. 1 mL of DNS reagent was accurately added to each tube, and the mixture was heated in a boiling water bath for 5 min, cooled under running water, and the absorbance was measured at 540 nm. An N-acetylglucosamine standard curve was established.

[0071] 3. Determination of the activity of Chi1 enzyme, which can efficiently degrade powder particles.

[0072] The enzyme activity of recombinant enzyme Chi1 was determined using 10 g / L colloidal chitin and powdered chitin as substrates. 100 μL of substrate was added to 850 μL of PBS (pH 7.0, 50 mM), preheated for 10 min, and then 50 μL of the crude enzyme solution obtained in step 9 of Example 3 was added. After reacting for 30 min, the mixture was transferred to a boiling water bath and boiled for 5 min to inactivate the enzyme. Then, 1 mL of DNS solution was added, and the mixture was boiled at 100 °C for 5 min. After cooling to room temperature, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was measured at 540 nm. An equal volume of inactivated enzyme solution was used as a blank control.

[0073] The enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar per minute at 37 °C.

[0074] Table 1 shows the specific enzyme activity assays for Chi1-degraded colloidal chitin and particulate chitin.

[0075] Example 5

[0076] Study on the enzymatic properties of Chi1 that can efficiently degrade powder particles

[0077] The enzymatic properties of Chi1, the highly efficient degradable chitinase for powder obtained in Example 3, were determined, including optimal temperature, temperature stability, optimal pH, pH stability, and the effect of metal ions on enzyme activity, using colloidal chitin as a substrate.

[0078] (1) Enzyme activity assay

[0079] See Example 4 for details.

[0080] (2) Effect of temperature on chitin endonuclease Chi1

[0081] ①Optimal reaction temperature

[0082] Under conditions of pH 7.0 (PBS buffer, 50 mM), using 10 g / L colloidal chitin as a substrate, the reaction system contained 100 μL colloidal chitin, 850 μL PBS buffer (pH 7.0, 50 mM), and 50 μL crude chitin endonuclease Chi1. The reaction was carried out for 30 min at temperature gradients of 25 ℃, 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, and 70 ℃. After the reaction, 1 mL of DNS reagent was immediately added to each reaction system, and the mixture was boiled for 5 min. The absorbance was then measured using a 540 nm UV-Vis spectrophotometer to determine the Chi1 enzyme activity at different temperatures. Based on the relative activity curves of the enzyme at different temperatures, the optimal reaction temperature of the Chi1 enzyme was determined. Figure 2 As shown in (a), the optimal reaction temperature for the chitin endonuclease Chi1 is 40 °C.

[0083] ② The thermal stability of the chitinase Chi1, which can efficiently degrade powder particles.

[0084] The crude enzyme solution of Chi1, which can efficiently degrade powder particles, was incubated at different temperatures (30 ℃-55 ℃, with an interval of 5 ℃) for 2 h, cooled in an ice-water bath for 10 min, and then the residual enzyme activity was detected and compared with the untreated enzyme activity to calculate the relative activity.

[0085] The results are as follows Figure 2 As shown in (b), the chitinase Chi1, which can efficiently degrade powder particles, is active at temperatures ranging from 30 to 55 ℃, exhibits good thermal stability at 30 to 40 ℃, and maintains an activity of over 80%.

[0086] 2. Effect of pH on chitinase Chi1

[0087] ①Optimal pH

[0088] The enzyme activity reaction temperature was 40 °C. Using 10 g / L colloidal chitin as the substrate, the enzyme activity was measured in 10 mmol / L buffer solutions (pH 3.0–6.0 sodium citrate buffer, pH 6.0–8.0 phosphate buffer, pH 9.0–11.0 glycine-sodium hydroxide buffer) with pH values ​​of 3–11. The optimal reaction pH value of the enzyme was determined by plotting the relative activity curves of the enzyme at different pH values.

[0089] The results are as follows Figure 3 As shown in (a), the optimal pH for the chitin endonuclease Chi1 is 7.0.

[0090] ② pH stability of chitosan Chi1

[0091] The enzyme solution was placed in 10 mmol / L buffer solutions of different pH values ​​(pH 3.0–6.0 sodium citrate buffer, pH 6.0–8.0 phosphate buffer, pH 9.0–11.0 glycine-sodium hydroxide buffer), and placed on ice for 2 h. The residual enzyme activity was then measured at the optimal reaction temperature and compared with the activity of the untreated enzyme to calculate the relative activity.

[0092] The results are as follows Figure 3 As shown in (b), the chitin endonuclease Chi1 exhibits activity at pH values ​​ranging from 3.0 to 11.0, with higher activity between pH values ​​of 4.0 and 10.0, and optimal activity at pH 7.0. Stability experiments indicate that the activity of chitin endonuclease Chi1 remains above 80% after incubation for 2 hours at pH values ​​ranging from 4.0 to 10.0.

[0093] 3. Effects of metal ions on chitinase Chi1

[0094] EDTA, MnCl2, CuCl2, NaCl, ZnCl2, CoCl2, FeCl2, NH4Cl, BaCl, CaCl2, LiCl, KCl, MgCl, and AlCl3 were added to the reaction system to a final concentration of 10 mmol / L, respectively. The enzyme activity was then measured under standard conditions and compared with the activity of the enzyme without any added metal ions to calculate the relative enzyme activity.

[0095] The results are as follows Figure 4 As shown, Mg 2+ Ba 2+ Na + Ca 2+ and Li + It has an activating effect on Chi1 enzyme activity, while 10 mM EDTA has no significant effect on enzyme activity. Example 6

[0096] Analysis of products from the hydrolysis of colloidal chitin by chitinase Chi1

[0097] (1) Sampling: Add 100 µL Chi1 enzyme solution, 400 µL colloidal chitin, and 1.5 mL PBS (pH 7.0, 50 mM) buffer to the system and react at the optimal temperature of 40 °C for 6 h. Take 300 μL samples at 0 h, 1 h, 3 h, and 6 h of the reaction, inactivate at high temperature, and freeze at -20 °C.

[0098] (2) Sample preparation: After thawing the frozen product, centrifuge it at 12000 r / min for 3 min in an Eppendoff AG 22331 Hamburg. Use a disposable syringe and a 0.22 μm water membrane to filter the product, reducing the generation of impurity peaks.

[0099] (3) Product Detection and Analysis: Enzymatic products were analyzed using a Shimadzu high-performance liquid chromatograph with a PrevailCarbohydrate ES 5u column (250 mm x 4.6 mm, Alltech Technology Ltd). Ultrasonicated acetonitrile and ultrapure water were used as the mobile phase. Detection method: The mobile phase was 75% acetonitrile and 25% ultrapure water. The injection volume was 5 μL, the run time was 22 min, the column temperature was 40 ℃, and the UV signal was 210 nm. The system was rinsed using the above mobile phase. After rinsing, the flow rate was increased to 1 mL / min for detection. After detection, the column was protected with 90% methanol.

[0100] Test results as follows Figure 5 As shown, the main product of the chitinase Chi1, which can efficiently degrade powder particles, hydrolyzes colloidal chitin, is (GlcNAc)2, with a small amount of GlcNAc also produced. Example 7

[0101] Analysis of the hydrolysis mode of chitinase Chi1

[0102] (1) Sampling: Take 10 μl of each of (GlcNAc)2, (GlcNAc)4, and (GlcNAc)6 chitosan oligosaccharides, add 10 μl of Chi1 enzyme solution to each, and react at the optimal temperature of 40 ℃ and 200 rpm. Take 2 μl of (GlcNAc)2 every 30 min, 2 h, 6 h, and 12 h. Take 2 μl of (GlcNAc)4 and (GlcNAc)6 at 5 min, 15 min, 30 min, and 1 h. Inactivate the enzymes at high temperature and store at -20 ℃.

[0103] (2) Sample dilution: After centrifugation, the obtained sample is diluted 10 times with ultrapure water and placed in a liquid chromatography vial for later use.

[0104] Product detection and analysis: Using high-performance liquid chromatography (HPLC), with filtered and sonicated acetonitrile and water as the mobile phase, after rinsing the tubing and column, the diluted sample was analyzed at a rate of 1 ml / min using 75% acetonitrile and 25% water. See Example 6 for details.

[0105] The results are shown in Figure 6As can be seen from Figures 6(a)-6(c), Chi1 can degrade chitobiose into monosaccharides over a relatively long period of time, and can degrade chitotetraose into disaccharides and trisaccharides in a short period of time. It can also degrade chitohexaose into disaccharides, trisaccharides, and tetrasaccharides, and finally all of them become disaccharides. It is a typical chitin endonuclease. Example 8

[0106] Chitinase Chi1 degrades particulate chitin

[0107] 100 µL of 10 g / L Chi1 enzyme solution, 10 g / L of chitin powder, and 900 µL of PBS (pH 7.0, 50 mM) buffer were added to the system. The reaction was carried out at the optimum temperature of 40 °C for 30 min. The amount of reducing sugar was measured by DNS, and the specific enzyme activity was calculated. According to Example 4, the enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar per minute at 37 °C, and the specific enzyme activity (U / mg) is enzyme activity / protein amount. The specific enzyme activity of chitinase Chi1 hydrolyzing chitin powder was calculated to be 91.76 U / mg. Example 9

[0108] Chitinase Chi1 degrades chitinous biomass

[0109] Add 100 µL of 10 g / L Chi1 enzyme solution, 100 g / L of lobster shell powder (collected from Nanjing Aquatic Products Market), hairy crab shell powder (Nanjing Vegetable Market), swimming crab powder (collected from Nanjing Aquatic Products Market), Ganoderma lucidum spore wall powder (purchased from Hangzhou Shouxian Valley Company), and Aspergillus niger mycelium powder (purchased from COFCO Company) to the system, add 900 µL of PBS (pH 7.0, 50 mM) buffer, and react at the optimum temperature of 40 ℃ for 6 h. The amount of reducing sugar produced is measured by DNS.

[0110] The results were calculated using the DNS standard curve and are shown in Table 2.

[0111] Table 2 shows the determination of reducing sugars produced by Chi1 degradation of chitin-based biomass.

[0112]

[0113] As shown in Table 2, the chitinase Chi1, which can efficiently degrade powder particles, can degrade chitinous biomass, and has the best effect on degrading lobster shells.

[0114] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. The application of Chitinase Chi1, a chitinase capable of efficiently degrading particulate chitin, in the degradation of colloidal chitin and particulate chitin, characterized in that, The amino acid sequence of the chitinase Chi1 is shown in SEQ ID NO.

2. When chitinase Chi1 degrades colloidal chitin and powdered chitin, the enzyme activity reaches 493.73 U / mg and 91.76 U / mg, respectively. The hydrolysis product is mainly (GlcNAc)2, with a small amount of GlcNAc and (GlcNAc)3.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the chitinase Chi1, which encodes chitinase Chi1, which can efficiently degrade chitin in powder particles, is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The optimal temperature and pH for enzymatic hydrolysis in this application are 40°C and 7.0, respectively.

4. The application according to claim 3, characterized in that, Mg in the application 2+ Ba 2+ Na + Ca 2+ and Li + It promotes the activity of chitinase Chi1 during enzymatic hydrolysis.

5. The application according to claim 1, characterized in that, The concentration of colloidal chitin or powdered chitin is 5-100 g / L.

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