A thermoresistant chitinase and its application

Abstract

This invention relates to the field of biotechnology, specifically to a chitinase and its applications. The chitinase of this invention has an amino acid sequence selected from either (A1) or (A2): (A1) The amino acid sequence is shown in SEQ ID NO:1; (A2) A protein with the same function as the protein shown in (A1) obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1. The chitinase obtained by this invention has an optimal temperature of 80℃, good thermal stability, and high hydrolysis efficiency, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a heat-resistant chitinase and its applications. Background Technology

[0002] Chitin is the second most abundant renewable natural polymer on Earth after cellulose, mainly distributed in the shells of crustaceans, the cuticles of invertebrates, the exoskeletons of insects, and the cell walls of fungi. Chitin has a large molecular weight, a highly ordered structure with numerous hydrogen bonds, and a high degree of crystallinity, making it insoluble in water, which limits its applications. Maximizing chitin's application value requires degrading it into lower molecular weight, water-soluble chitosan oligosaccharides. Traditional chitin degradation relies on chemical methods; industrially, concentrated hydrochloric acid is generally used to degrade chitin into monosaccharides. Higher hydrochloric acid concentrations and reaction temperatures result in faster reaction rates. However, this method struggles to produce polysaccharides with high polymerization degrees. Furthermore, chemical degradation is energy-intensive and generates large amounts of polluted wastewater, posing a significant environmental threat. In contrast, the hydrolysis of chitin using chitinases offers milder reaction conditions and can directly hydrolyze crystalline chitin, making it both energy-efficient and environmentally friendly. Therefore, this method has broad industrial application prospects.

[0003] Chitin hydrolysis products and their derivatives possess biocompatibility and broad pharmacological activities, making them highly valuable in applications and economics. Chitosan oligosaccharides have been reported to prevent tumor growth, treat asthma, improve bone strength, prevent malaria, and can be used as gene delivery vectors in gene therapy. The biological activities of chitosan oligosaccharides include antibacterial, antiviral, antioxidant, immunomodulatory, blood pressure control, and cholesterol-lowering effects. Furthermore, chitosan hexasaccharides have been shown to possess even greater biological activities, including antibacterial, antitumor, and immune-enhancing effects.

[0004] To date, many chitinases have been identified and enzymatically characterized, such as those from *Serratia marcescens*, *Paenicibacillus barengoltzii*, *Corallococcus sp.*, *Drosera rotundifolia*, *Streptomyces alfalfae*, and *Streptomyces albolongus*. However, most of these are mesophilic chitinases, which cannot meet the demanding conditions of industrial applications. Thermophilic enzymes are a class of enzymes that retain high activity at temperatures ranging from 60°C to 125°C. Compared to mesophilic enzymes, thermophilic enzymes have unique advantages, such as high catalytic activity, good thermal stability, longer storage time (at room temperature), and good tolerance to organic solvents. Thermophilic enzymes are more stable and can be used for longer periods, thereby reducing the cost of enzymes in biotechnology. Furthermore, degrading polymers at high temperatures offers numerous advantages, such as decreased viscosity, increased diffusion coefficient, and increased solubility of organic matter as temperature rises, thereby improving reaction rates and reducing the risk of bacterial contamination. Therefore, identifying thermophilic enzymes with good thermal stability and high enzymatic activity is crucial for industrial applications.

[0005] Chitinases derived from thermophilic bacteria possess extremely high optimum temperatures and good thermal stability. For example, the optimum temperature for chitinase Tc-ChiD from *Thermococcus chitonophagus* is 90℃, and its half-life at 100℃ is 48 min. Chitinases ChiAΔ5 and ChiAΔ4 from *Thermococcus kodakaraensis* KOD1 have optimum temperatures of 85℃ and 90℃, respectively, with ChiAΔ4 exhibiting a half-life exceeding 7 h at 100℃. Although these thermophilic chitinases possess good thermal stability, their enzyme activity and catalytic efficiency are quite low. Summary of the Invention

[0006] To address the shortcomings of existing methods, the present invention aims to provide a highly active, thermostable chitinase and its applications. The chitinase ActChi of the present invention has an optimal reaction temperature of 80°C and maintains high activity even at high temperatures, thus it is a thermophilic enzyme.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] Using metatranscriptomics, we discovered a chitinase, ActChi, with high transcriptional levels during the high-temperature stage of composting. This chitinase consists of a malectin domain, an Fn3 domain, and a catalytic structure (CD). chiIt is composed of a domain. The chitinase ActChi (full length) contains 624 amino acids.

[0009] Specifically, the chitinase ActChi has an amino acid sequence selected from the following (A1) or (A2):

[0010] (A1) The amino acid sequence is shown in SEQ ID NO:1;

[0011] (A2) A protein having the same function as the protein shown in (A1) obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in SEQ ID NO: 1.

[0012] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of this invention using known methods, such as directed evolution or point mutation. Artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the isolated protein of this invention, provided they encode and function a protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0013] As a preferred technical solution of this application, the chitinase ActChi is derived from actinomycetes.

[0014] The present invention also protects biological materials, which may be any one of the following (B1)-(B4): (B1) DNA molecules encoding chitinase ActChi;

[0015] (B2) An expression cassette containing the DNA molecule described in (B1);

[0016] (B3) A recombinant vector containing the DNA molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2);

[0017] (B4) A recombinant microorganism containing the DNA molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B3).

[0018] As a preferred technical solution of this application, in (B1), the DNA molecule is selected from the following (C1) or (C2):

[0019] (C1) Nucleotide sequence is shown in SEQ ID NO:2;

[0020] (C2) A DNA molecule that has at least 80%, 85%, 90%, 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein with the same function.

[0021] The vectors described in this invention are known to those skilled in the art, including but not limited to: plasmids, bacteriophages, viral vectors, etc., such as the vector pCold-TF.

[0022] The microorganisms described in this invention may be yeast, bacteria, or fungi, etc.

[0023] This invention also protects a chitinase mutant Fn3-CD chi The chitinase ActChi, mentioned above, is obtained by deleting malectin.

[0024] Preferably, the Fn3-CD chi The amino acid sequence is shown in SEQ ID NO: 3.

[0025] This invention also protects a chitinase mutant CD chi The chitinase ActChi described above is obtained by deleting malectin and Fn3.

[0026] Preferably, the CD chi The amino acid sequence is shown in SEQ ID NO: 4.

[0027] This invention also protects a chitinase fused with ChBD-CD. chi The fused chitinase ChBD-CD chi The amino acid sequence is shown in SEQ ID NO: 6.

[0028] The fused chitinase ChBD-CD chi The hydrolytic activity against crystalline chitin was increased by 4 times.

[0029] This invention also protects a method for improving the hydrolytic activity of the chitinase described above on crystalline chitin, by synthesizing the chitin-binding domain ChBD into the ActChi catalytic domain CD of the chitinase described in claim 1. chi At the N-terminus, a chitinase fused to ChBD-CD was obtained. chi .

[0030] Specifically, the amino acid sequence of the chitin-binding domain ChBD is shown in SEQ ID NO: 5.

[0031] Specifically, the fused chitinase ChBD-CD chi The amino acid sequence is shown in SEQ ID NO: 6.

[0032] The present invention also protects the DNA molecule encoding the chitinase fused above.

[0033] This invention also protects expression cassettes containing the aforementioned DNA molecules, recombinant vectors, and recombinant microorganisms.

[0034] This invention also protects the chitinase mutant Fn3-CD described above. chi The chitinase mutant CD as described in claim 4 chi The application of the fused chitinase of claim 6 or 7, the DNA molecule of claim 8, the expression cassette containing the DNA molecule of claim 8, the recombinant vector, the recombinant microorganism, in the hydrolysis of chitin.

[0035] Beneficial effects

[0036] The purpose of this invention is to provide a chitinase and its applications, which, compared with the prior art, has the following beneficial effects:

[0037] (1) The chitinase ActChi of the present invention has an optimal reaction temperature of 80°C and can maintain high activity at high temperature, and is a thermophilic enzyme.

[0038] (2) This invention is carried out via CD chi Adding a chitin-binding domain (ChBD) to the N-terminus yields ChBD-CD. chi Its hydrolytic activity against crystalline chitin was increased by 4 times. Attached Figure Description

[0039] Figure 1 Example 1: ActChi, Fn3-CD chi and CD chi The structural domain positions: malectin domain (residues 14-150, magenta), Fn3 domain (residues 174-258, brown), and the catalytic domain of GH18 chitinase;

[0040] Figure 2 Phylogenetic tree analysis of ActChi in Example 1;

[0041] Figure 3 The sequence alignment of the catalytic domain of ActChi in Example 1 with other GH18 chitinases is shown. The listed sequences include chitinases from Actinoma durageliboluensis (WP_138632822.1), Streptomyces aurantiacus (WP_106968041.1), Chitiniphilus shinanonensis (BAK53954.1), and Chloroflexibacterium (RMG87951.1). Black triangles indicate conserved amino acid residues;

[0042] Figure 4 Example 1 (A) Overall structure of ActChi; malectin domain, magenta; Fn3 domain, brown; catalytic domain, cyan; (B) Homology model of the ActChi catalytic domain; chitin insertion domain (CID), magenta; three pairs of disulfide bonds (C322-C399, C425-C429 and C481-C495), yellow; catalytic motif, red;

[0043] Figure 5 Example 1: SDS-PAGE analysis of chitinase purification results; Lane M: marker; Lane 1: CD chi Lane 2: Fn3-CD chi Lane 3: ActChi;

[0044] Figure 6 The optimal reaction conditions for Example 1 are: (A) optimal pH; (B) optimal temperature.

[0045] Figure 7 The following are the thermal inactivation properties of chitinase in Example 1: (A) Thermal stability at 80°C; (B) Thermal stability at 85°C; (C) First-order inactivation diagram at 80°C; (D) First-order inactivation diagram at 85°C.

[0046] Figure 8 Example 1: HPLC analysis of chitinase hydrolysis products. (A)Fn3-CD chi Hydrolysate analysis; (B)CD chi Hydrolysis analysis of (C) ActChi;

[0047] Figure 9 This is a schematic diagram of the structure of chitinase fused in Example 2;

[0048] Figure 10 Example 2 ChBD-CD chi The results of homology modeling;

[0049] Figure 11 Example 2: SDS-PAGE analysis of the purification results of the chitinase fusion; Lane M: marker; Lane 1: ChBD-CD chi ;

[0050] Figure 12 Example 2: Optimal reaction conditions; (A) Optimal pH; (B) Optimal temperature;

[0051] Figure 13 The thermally inactive properties of the fused chitinase in Example 2; (A) thermal stability of the fused chitinase at 80°C; (B) thermal stability of the fused chitinase at 85°C. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0053] The amino acid sequence of the chitinase ActChi was obtained from the metatranscriptome database of high-temperature compost samples from the research group of Professor Liu Dongyang at the College of Resources and Environmental Protection, Nanjing Agricultural University. The genes for ActChi and two truncated proteins (Fn3-CD) were also obtained. chi and CD chi It was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0054] Plasmid cloning strain Escherichia coli DH5α (hereinafter referred to as DH5α) and expression strain Escherichiacoli Rosetta TM (DE3) (hereinafter referred to as Rosetta) was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0055] The expression plasmid pCold-TF was provided by our laboratory; this vector contains a 10×His tag, a TF lysis tag, and a TEV cleavage site (sequence: ENLYFQS) at the N-terminus, which facilitates protein purification.

[0056] Example 1: Obtaining chitinase ActChi and its mutants

[0057] 1. Bioinformatics Analysis

[0058] Using macrotranscriptional analysis, the transcriptional level of glycoside hydrolases in compost was analyzed. A chitinase with a high transcriptional level during the high-temperature period of composting was identified and named ActChi.

[0059] The chitinase ActChi (full-length) contains 624 amino acids. The amino acid sequence of ActChi was analyzed using the PSI-BLAST web server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Domain positional features of ActChi were plotted using DOG 2.0 software. The amino acid sequences of 17 GH18 family chitinases were aligned using the muscle program in MegaX, and a neighbor-joining developmental tree was constructed with 1000 bootstrap values. Sequence alignment was performed using Clustalomega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ), and the alignment results were plotted using ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / ESPript / index.php). This study used ProtParam (https: / / web.expasy.org / protparam / ) to calculate the theoretical molecular weight (MW) and molar extinction coefficient, and used AlphaFold2 to study the chitinases ActChi and ChBD-CD. chi Three-dimensional structure prediction was performed, and the protein structure was visualized and analyzed using PyMOL software.

[0060] 2 Gene Synthesis

[0061] The full-length chitinase gene ActChi and the truncated protein gene Fn3-CD were used. chi (Only delete the malectin structure field) and CD chi The amino acid sequence (with both malectin and Fn3 domains deleted) was optimized by Beijing Qingke Biotechnology Co., Ltd. according to the codon preference of *E. coli*. The genes of ActChi and its two truncated mutants were then cloned into the pCold-TF expression vector. This vector is a cold shock expression vector, which can improve the yield, purity, and solubility of recombinant protein expression in cells. The pCold-TF vector stored in the laboratory has been modified by adding a TEV restriction site (ENLYFQS) at the N-terminus to facilitate protein purification.

[0062] 3. Recombinant plasmid transformation

[0063] The recombinant plasmid was transformed into Rosetta cells, an E. coli expression competent cell line, to prepare for protein expression and purification.

[0064] First, Rosetta competent cells were thawed on ice. 5-10 μL of recombinant plasmid (approximately 100 ng / μL) was added to 100 μL of competent cells, slowly mixed, and incubated on ice for 25 min. Then, the cells were heat-shocked in a 42°C water bath for 45 s, followed immediately by an ice bath for 2 min. In a clean bench, 500 μL of antibiotic-free LB broth was added to the competent cells infused with the recombinant plasmid, and the cells were cultured at 37°C with shaking for 60 min. The bacterial culture was then evenly spread onto LB agar plates containing ampicillin and incubated overnight at 37°C.

[0065] 4. Protein Expression and Purification

[0066] 4.1 Protein Expression

[0067] A single colony was picked from an LB solid agar plate and inoculated into 50 mL of LB liquid agar (ampicillin final concentration 50 μg / mL). The culture was incubated at 37°C in a constant temperature shaking incubator until the bacterial OD reached its maximum. 600 Approximately 0.5. Take 20 mL of bacterial culture and inoculate it into 1 L of LB liquid medium (containing 50 μg / mL ampicillin), and incubate at 37℃ and 200 rpm. Detect the OD value of the bacterial culture. 600 When the value reached about 0.6, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 24 h at 15℃ and 150 rpm.

[0068] 4.2 Protein purification

[0069] The target protein was purified using a Ni affinity chromatography column. The purification process is as follows:

[0070] (1) Centrifuge the bacterial culture induced for 24 h at 4 °C at 8000 × g for 15 min and collect the cell pellet. Add lysis buffer to the cell pellet at a ratio of 1:10 (m / v) and mix well to ensure that there are no bacterial clumps in the resuspended pellet.

[0071] (2) The resuspended cell solution was disrupted using a high-pressure homogenizer: the pressure was slowly increased to 750 bar at a disruption temperature of 4℃ until the bacterial solution was clear and transparent. The disrupted bacterial solution was centrifuged at 15000×g at 4℃ for 60 min and the supernatant was collected.

[0072] (3) Equilibrate the Ni affinity chromatography column with the first purification equilibration buffer. When the inflow of equilibration buffer is 8 times the column volume, reduce the flow rate to 1 mL / min and start loading the sample slowly. Then, use 5 times the column volume of washing buffer to elute the impurities. After that, use elution buffer to elute the target protein (at this time, the protein has a soluble tag TF at the N-terminus) and collect it.

[0073] (4) In order to remove the soluble tag (TF), TEV protease (1:100, m / m) was added to the collected protein eluent and digested overnight in protein dialysate (I).

[0074] (5) Equilibrate the Ni affinity chromatography column to 8 column volumes using the second purification equilibration buffer, load the enzyme digested mixture onto the column, and collect the eluent target protein solution during this process. Dialyze the protein obtained from the second purification overnight in protein dialyzing buffer (II).

[0075] (6) The concentration of the target protein was detected using a micro-detector. The principle is that the absorption peak of the protein at a wavelength of 280 nm is determined by aromatic residues. Therefore, the extinction coefficient of the protein can be predicted by the proportion of tryptophan in the protein amino acids, and then the concentration of the protein in the solution can be calculated.

[0076] 4.3 SDS-PAGE Analysis

[0077] (1) Install the glue-making plate and check for leaks. Add the prepared 10% separating glue to the top 1 / 4 of the glue plate gaps, seal the gaps with water, and let it stand. After the separating glue has solidified, pour off the water that was used to seal it, add the prepared 5% concentrated glue, insert the corresponding comb, and let it stand.

[0078] (2) Sample preparation: Mix the protein to be tested with β-mercaptoethanol at a ratio of 20:1, and add sample buffer to the mixture at a ratio of 1:1. After mixing, heat denature at 95°C for 4 min.

[0079] (3) Sample loading: Place the solidified gel into the electrophoresis tank, add an appropriate amount of electrode buffer, and slowly pull out the comb. Add 4 μL of sample to each well.

[0080] (4) Electrophoresis: Sample concentration was performed at 85V and protein separation was performed at 185V.

[0081] (5) Staining and destaining: The gel was rapidly stained with Coomassie Brilliant Blue rapid staining solution. The gel was then destaining in heated deionized water.

[0082] 5. Characterization of Enzymatic Properties

[0083] 5.1 Creating a Standard Curve

[0084] Solutions of different concentrations were prepared using a 1 mg / mL N-acetyl-D-glucosamine standard solution. After reacting with DNS, the absorbance was measured at 540 nm using a spectrophotometer. A standard curve was plotted with the N-acetyl-D-glucosamine content as the ordinate and the absorbance value as the abscissa (formula: y = 0.4055x - 0.0038R). 2 =0.9936).

[0085] 5.2 Chitinase Activity Assay Method

[0086] Chitinase activity was determined using the DNS method. The reaction mixture consisted of 0.1 mL of appropriately diluted enzyme solution, 0.5% colloidal chitin, and 50 mM acetate-sodium acetate buffer (pH 6.0), and was reacted at 80 °C for 30 min. The reaction was terminated by adding 1 mL of DNS reagent, followed by boiling in a water bath for 10 min, cooling, and centrifugation at 8000 × g for 5 min. Finally, the absorbance of the supernatant was measured at 540 nm. Chitinase activity was defined as the amount of enzyme required to produce 1 μmol of N-acetyl-D-glucosamine per minute.

[0087] 5.3 Determination of Optimal pH and Optimal Temperature

[0088] Using 0.5% colloidal chitin as a substrate, the optimal pH of chitinase was determined by detecting the residual activity of chitinase in different 50 mM buffer solutions (pH 2–11) at 80 °C. The buffer solutions used were: Gly-HCl buffer (pH 2.0–4.0), acetate-sodium acetate buffer (pH 4.0–6.0), phosphate buffer (pH 6.0–7.0), Tris-HCl buffer (pH 8.0–9.0), and Gly-NaOH buffer (pH 9.0–11.0).

[0089] A 0.5% colloidal chitin solution was prepared using an acetate-sodium acetate buffer solution with a pH of 6. The residual enzyme activity of chitinase at different temperatures was detected using the enzyme activity assay method described in section 5.2 within the range of 40-95℃, thereby determining the optimal temperature for chitinase.

[0090] 5.4 Determination of thermal stability and half-life

[0091] To investigate the thermostability of chitinase, chitinase was incubated at different temperatures (80, 85℃) for different times (5, 10, 15, 20, 25, 30, 35 min), and equal volumes of samples were recovered and cooled on ice for 5 min. Residual enzyme activity was measured under optimal pH and temperature conditions. The thermodynamics of chitinase deactivation is considered to conform to the first-order deactivation equation, which can be written as:

[0092] lnU1-lnU0=-kt

[0093] t = ln2 / k

[0094] k: Inactivation rate constant, obtained by calculating the slope of the equation; U1: Chitinase activity after heat incubation; U0: Untreated chitinase activity; t: Half-life.

[0095] Using ln(U1 / U0) as the ordinate and time as the abscissa, the value of k (inactivation constant) was obtained by linear fitting using Origin software, and then the half-life of chitinase at this temperature was obtained.

[0096] 5.5 Activity and enzyme kinetic parameters of different chitins

[0097] The purified chitinase was reacted with various substrates at 80°C in 50 mM acetate-sodium acetate buffer (pH 6.0) for 30 min, and the substrate specificity of the chitinase was then determined using the enzyme activity assay method described in section 5.2. The substrates tested (0.5%, w / v) were colloidal chitin and crystalline chitin (α-chitin and β-chitin).

[0098] Chitinase was reacted with different concentrations of colloidal chitin (1-35 mg / mL) in 50 mM acetate-sodium acetate buffer (pH 6.0), and its activity was determined by measuring the reaction time at 80°C for 5 min. Enzyme kinetic parameter V max With K m The result was obtained by fitting the data using the Michaelis-Menten function in Origin 9.0 software.

[0099] 5.6 Analysis of Hydrolysis Products

[0100] The hydrolysis products of chitinase were analyzed using high-performance liquid chromatography (HPLC). Purified chitinase was reacted with 1% colloidal chitin under optimal conditions for different times (15, 30, 60, 120 min), then the reaction was terminated in a boiling water bath and the supernatant was filtered (pore size 0.22 μm). HPLC analysis was performed using a Shodex Asahipak NH2P-50 4E column (250 × 4.6 mm) (Shimadzu-LC-20A, Tokyo, Japan) with an injection volume of 10 μL and a mobile phase of 70% acetonitrile. The hydrolysis products were detected using an evaporative light detection (ELSD) detector at 35 °C and a flow rate of 1 mL / min. Chitin oligomers of DP(1–7) were used as standards.

[0101] result

[0102] 1. Bioinformatics Analysis

[0103] PSI-Blast analysis was used to analyze chitinases exhibiting high transcriptional levels during the high-temperature phase of composting. The results indicated that this 637-amino acid protein belongs to the GH18 family of chitinases. Figure 1As shown, the enzyme contains an N-terminal malectin domain (residues 14-150), a fibronectin III domain (Fn3) (residues 174-258), and a C-terminal GH18 chitinase catalytic domain (CD). chi (residues 270-622).

[0104] The phylogenetic tree of ActChi was constructed using MegaX, and the results are as follows: Figure 2 As shown, this chitinase belongs to the A subfamily of chitinases in the GH18 family, and we name this chitinase ActChi.

[0105] Multiple sequence alignment was performed with chitinases from other microbial sources, and the results were as follows: Figure 3 As shown, the catalytic domain of ActChi has a conserved DxDxE catalytic sequence, in which glutamate is the catalytic residue essential for activity, which is also a characteristic of GH18 family chitinases.

[0106] Homology modeling was performed on the full-length and catalytic domains of ActChi. The results are as follows: Figure 4 As shown, the catalytic domain is a (β / α)8 barrel-shaped structure composed of 8 β-strands and 8 α-helices. On the 7th β-strand, there is a chitin insertion domain (CID) consisting of 5 antiparallel β-strands and one α-helice, which is characteristic of the GH18 chitinase A subfamily. Based on phylogenetic tree and homology modeling results, we consider this chitinase to be an exochitinase. The catalytic amino acid DxDxE is located in the middle of the substrate-binding cleft in this region. The catalytic domain contains three pairs of disulfide bonds, two pairs (C322-C399, C425-C429) located on the surface of the (β / α)8 barrel, and the other pair (C481-C495) located on the CID.

[0107] 2 Expression and Purification

[0108] To investigate the effects of malectin and Fn3 on the enzymatic properties of chitinase, we sequentially deleted these two domains to synthesize the truncated ActChi protein Fn3-CD. chi and CD chi ,like Figure 5 As shown. Calculations indicate that the theoretical molecular masses of these three enzymes are 67 kDa, 50 kDa, and 40 kDa, respectively. The two truncated protein genes and ActChi were cloned into the pCold-TF expression vector, and then transformed into Rosetta(DE3) competent cells for induction and expression. The supernatant from cell lysis was purified by two Ni affinity chromatography analyses. The purity of the three proteins was determined using SDS-PAGE gel electrophoresis. Figure 3 As shown, all three proteins appear as a single band, with apparent molecular weights of approximately 67 kDa, 50 kDa, and 40 kDa, respectively.

[0109] 3. Enzymatic properties

[0110] The optimal temperature and pH of chitinase were determined using 0.5% colloidal chitin as a substrate. The results are as follows: Figure 6 As shown, all three chitinases exhibited peak activity at pH 6.0, and within the pH range of 4-7, all three showed residual activity exceeding 50%. However, the activities of all three chitinases decreased rapidly at pH ranges of 2-5 and above pH 7. These results indicate that malectin and Fn3 do not alter the effect of pH on chitinase activity. The optimal temperature for ActChi and its two truncated mutants is 80℃. Within the temperature range of 70-85℃, the residual activities of all three chitinases remained above 60%, but at 95℃, the residual activities decreased to approximately 20%. This indicates that malectin and Fn3 do not affect the optimal temperature for chitinase activity.

[0111] Regarding the thermal stability of chitinases, such as Figure 7 As shown in Table 1, the half-lives of ActChi at 80℃ and 85℃ are 8.5 min and 1.5 min, respectively. The enzyme activity decreases sharply to near zero after incubation at 85℃ for 10 min. chi Incubation at 80℃ for 30 minutes resulted in a slow decrease in residual enzyme activity to approximately 40%, significantly superior to ActChi, and exhibited similar thermal stability to ActChi at 85℃. chi The half-lives at 80℃ and 85℃ were 20.1 min and 1.8 min, respectively. Fn3-CD chi Its thermal stability at 80℃ is better than that of CD. chi It is even better, especially with a half-life of 13.3 min at 85℃. After incubation at 85℃ for 30 min, Fn3-CD... chi Approximately 20% of the enzyme activity remains. Fn3-CD chi Its thermal stability at 85℃ is significantly better than that of ActChi and CD. chi These results indicate that the malectin domain reduces the thermal stability of proteins, while the Fn3 domain can improve the thermal stability of chitinases.

[0112] Table 1 ActChi, Fn3-CD chi and CD chi Half-life at 80℃ and 85℃

[0113]

[0114] 4. Enzyme kinetic parameters

[0115] The experiment measured ActChi and Fn3-CD chi and CD chi The dynamic parameters of Fn3-CD are shown in Table 2. chi K m The value was 15.2 mg / mL, lower than CD. chi Interestingly, Fn3-CD chi Performance with CD chi Similar k cat The value is 446.8s. -1 And catalytic efficiency (k cat / K m The duration increased slightly to 29.5s. -1 / mg / mL. Of the three chitinases, ActChi's K... m The lowest concentration (14.6 mg / mL) exhibited the highest substrate affinity with Fn3-CD. chi Compared to catalytic efficiency (k cat / K m The efficiency was increased by 1.4 times. These results indicate that the malectin and Fn3 domains can enhance substrate affinity and improve the catalytic efficiency of chitinase.

[0116] Table 2 Enzyme kinetic parameters of ActChi and truncated proteins

[0117]

[0118] 5. Substrate specificity

[0119] All three chitinases exhibited the greatest hydrolytic activity against colloidal chitin. As shown in Table 3, ActChi and Fn3-CD... chi The hydrolytic activity in reaction with insoluble chitin (α-Chitin and β-chitin) is CD. chi The results indicate that the three enzymes exhibit strong substrate specificity, with colloidal chitin being the optimal substrate. The Fn3 and malectin domains can bind to chitin sugar chains distributed on the crystal surface, shortening the distance between the enzyme and the substrate, increasing the enzyme concentration on the substrate, and thus enhancing the activity of ActChi and Fn3-CD. chi Activity of hydrolyzing insoluble chitin.

[0120] Table 3 Hydrolytic activities of ActChi and truncated enzymes on different substrates.

[0121]

[0122] 6. Analysis of hydrolysis products

[0123] Chitinases ActChi and Fn3-CD were detected by HPLC. chi and CD chi The hydrolysis products. The results are as follows: Figure 8 As shown, the hydrolysis products of the three chitinases using colloidal chitin as a substrate were all chitobiose at different time points. These results indicate that malectin and the Fn3 domain cannot change the type of hydrolysis product. Chitobiose has high application value and can be used as a cosmetic ingredient, dietary supplement, and antioxidant.

[0124] Example 2: Construction of chitinase fusion

[0125] 1. Sequence of chitin-binding domain and construction of chitin fusion enzyme

[0126] The chitin-binding domain (ChDB3) from Pyrococcus furiosus was searched and selected from the NCBI database (https: / / www.ncbi.nlm.nih.gov): TTPVPVSGSLEVKVNDWGSGAEYDVTLNLDGQYDWTVKVKLAPGATVGSFWSANKQEGNGYVIFTPVSWNKGPTATFGFIVNGPQGDKVEEITLEINGQVIDIWTPTGGTTPTPTTTTTSTPTPSQTPTPTPTPTPTPTPTPTLTPTPLPGNANPIPEH

[0127] Based on the original position of the chitin-binding domain in glycoside hydrolases, it was fused into CD4, which only contains the catalytic domain. chi The N-terminus of the chitin-binding domain. The synthesis and fusion of the chitin-binding domain were completed by General Biotechnology (Anhui) Co., Ltd.

[0128] 2 Homology modeling of chitinase fusion

[0129] Using AlphaFold2 to target chitinase ChBD-CD chi The three-dimensional structure of the catalytic domain was predicted, and the protein structure was visualized using PyMOL software.

[0130] 3 Expression and Purification

[0131] The chitinase fusion was expressed and purified, following the steps outlined in Example 1.

[0132] 4. Characterization of Enzymatic Properties

[0133] The enzymatic properties of the fused chitinase were detected, and the experimental procedures were as described in Example 1.

[0134] result

[0135] 1. Construction of fusion chitinase

[0136] We selected a chitin-binding domain (ChBD) from the NCBI database and, based on its original position in the glycoside hydrolase, fused it into the catalytic domain (CD) of ActChi. chi The N-terminus of the ) . The location of the structural domain is as follows Figure 9 As shown. ChBD-CD chi The molecular weight is 56.7 kDa, and the amino acid sequence is shown in SEQ ID NO: 6.

[0137] 2 Homologous models of chitinase fusion

[0138] ChBD-CD chi Homology modeling was performed, and the results are as follows: Figure 10 As shown, ChBD consists of two quadruple β-sheets, exhibiting a typical β-sandwich structure, similar to other carbohydrate-binding module 2 family proteins. The chitin-binding surface is flat, composed of three Trp residues (Trp17, Trp51, and Trp69) exposed to the solvent. Hydrophobic interactions play a dominant role in the recognition of crystalline chitin, and ChBD demonstrates higher substrate specificity for chitin compared to cellulose. ChBD is located within the catalytic domain CD. chi At the N-terminus, since ActChi is a continuously hydrolyzing exochitinase, ChBD helps to adsorb and guide the chitin chain into the enzyme catalytic channel.

[0139] 3 Expression and purification of fusion chitinase

[0140] The successfully synthesized and cloned recombinant plasmid fused with chitinase was transformed into *E. coli* Rossetta (DE3) competent cells for induced expression. The protein was obtained after two purifications using Ni affinity chromatography. The purity of the purified protein was assessed using SDS-PAGE gel chromatography. Results are as follows: Figure 11 As shown, the chitinase fusion band is a single band, indicating high protein purity, and the apparent molecular weight is consistent with the prediction results of the online server ProtParam.

[0141] 4. Effects of pH and temperature on fusion chitinase

[0142] The effects of pH and temperature on the fusion chitinase were investigated. The results are as follows: Figure 12 As shown, ChBD-CD chi The optimal pH and temperature are the same as ActChi, at pH 6.0 and 80℃, respectively. The activities of the chitinase fused with ChBD and the wild-type chitinase decrease rapidly on both sides of the optimal pH. However, at 80-90℃, the activities of ChBD-CD...chi The relative enzyme activity is relatively high. These results indicate that fusing heterologous ChBD cannot change the optimal pH and optimal temperature of chitinase, but it can improve the activity of chitinase at high temperatures.

[0143] 5. Thermal stability of fused chitinase

[0144] We incubated the chitinase fused with ChBD at 80°C and 85°C for different times and measured the residual enzyme activity to determine the thermostability and half-life of the fused chitinase. The results are as follows: Figure 13 As shown in Table 4, the thermal inactivation trend of the fused chitinase at 80℃ or 85℃ is similar to that of the chitinase ActChi: at 80℃, it slowly inactivates within 30 min; at 85℃, the chitinase activity decreases rapidly within 10 min. However, ChBD-CD chi The half-life at 80℃ and 85℃ decreased by 2.5 and 1.5 times that of ActChi, respectively. These results indicate that fusing ChBD derived from thermophilic bacteria to the N-terminus of the chitinase catalytic domain significantly improves the thermostability of chitinase.

[0145] Table 4 Half-life of chitinase fusion

[0146]

[0147]

[0148] Comparison of the hydrolytic activity of 6 fused chitinases on crystalline chitin

[0149] Using 0.5% crystalline chitin as a substrate, the hydrolytic activity of the fusion chitinase on crystalline chitin was determined using the standard enzyme activity assay method described above. The results are shown in Table 5. Compared to ActChi, ChBD-CD... chi The hydrolytic activity was increased by 4 times. Based on the above results, we believe that ChBD fused with CBM family 2 (CBM2) can significantly improve the hydrolytic activity of chitinase on crystalline chitin.

[0150] Table 5 Comparison of the hydrolytic activity of fused chitinase on crystalline chitin.

[0151]

[0152] In summary, the experiment in Example 2 was conducted using CD. chi By fusing chitin-binding domains (ChBDs) at both ends, the chitinase ChBD-CD was obtained. chi The enzymatic properties of the fused chitinase were studied. The optimal temperatures for the fusion chitinase were 80℃ and 6.0℃, respectively. ChBD-CD chiThe thermal stability at 80℃ and 85℃ is significantly improved compared to wild-type chitinase ActChi.

[0153] Additionally, ChBD-CD chi It has a 4-fold increased hydrolytic activity on crystalline chitin.

[0154] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A chitinase ActChi, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A biomaterial, wherein the biomaterial may be any one of the following (B1)-(B4): (B1) DNA molecule encoding chitinase ActChi; (B2) An expression cassette containing the DNA molecule described in (B1); (B3) A recombinant vector containing the DNA molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2); (B4) A recombinant microorganism containing the DNA molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B3); in, (B1) The nucleotide sequence of the DNA molecule is shown in SEQ ID NO:

2.

3. A method for improving the hydrolytic activity of chitinase on crystalline chitin according to claim 1, characterized in that, The chitin binding domain ChBD is synthesized to the N-terminal of the catalytic domain CD of the chitinase ActChi according to claim 1, obtaining a fusion chitinase ChBD-CD having the amino acid sequence shown in SEQ ID NO: 6 chi . The chitin binding domain ChBD is synthesized to the N-terminal of the catalytic domain CD of the chitinase ActChi according to claim 1, obtaining a fusion chitinase ChBD-CD having the amino acid sequence shown in SEQ ID NO: 6 chi .

4. A chitinase fused with ChBD-CD chi Its characteristics are, The fusion chitinase ChBD-CD chi The amino acid sequence of ChBD-CD is shown as SEQ ID NO:

6.

5. Encoding the chitinase ChBD-CD fusion enzyme as described in claim 4 chi DNA molecules.

6. An expression cassette containing the DNA molecule of claim 5.

7. A recombinant vector containing the DNA molecule of claim 5.

8. A recombinant microorganism containing the DNA molecule of claim 5.

9. The chitinase ActChi according to claim 1, the biomaterial according to claim 2, and the fusion chitinase ChBD-CD according to claim 4. chi The application of the DNA molecule of claim 5, the expression cassette of claim 6, the recombinant vector of claim 7, or the recombinant microorganism of claim 8 in the hydrolysis of chitin.

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