Endocellulase mutants and uses thereof

Abstract

The application provides an endocellulase mutant and application thereof, and belongs to the technical field of enzyme preparation.The mutant is an amino acid sequence after substitution mutation at any one or several of positions 105, 205 and 233 of the amino acid sequence shown in SEQ ID NO.1.The application adopts NDT codon and single-point mutation technology, combines Caver tunnel engineering guidance and Hotspot precise prediction technology, finds the influence of a key structure (tunnel) in the enzyme structure on the enzyme catalytic performance, and obtains a persistent endocellulase mutant with significantly improved enzyme activity.The mutant of the persistent endocellulase has high catalytic activity, and provides new feasibility for industrialized biological conversion of cellulose raw materials.

Description

Technical Field

[0001] This invention relates to the field of enzyme preparation technology, and in particular to an endonuclease mutant and its application. Background Technology

[0002] Cellulose is the most abundant renewable resource on Earth, and utilizing enzymes to degrade cellulose into fermentable sugars is one of the keys to realizing the resource utilization of cellulose. However, the current cellulose degradation process requires a wide variety of enzymes and large quantities, resulting in excessively high enzyme application costs. Enzyme preparation costs account for more than 30% of the total cost of cellulosic ethanol production, severely restricting the cellulose biorefining process and becoming a bottleneck factor for the commercialization of cellulosic ethanol and high-value chemicals.

[0003] Compared to conventional cellulose-degrading enzyme systems, continuously degrading cellulases discovered in recent years have demonstrated numerous advantages and hold immense potential for application in the low-cost biotransformation of cellulose. Continuously degrading cellulases are bifunctional, exhibiting characteristics of both endoglucanases and exoglucanases. They can continuously degrade cellulose by acting solely in synergy with β-glucosidases. Furthermore, unlike exoglucanases, continuously degrading cellulases lack a loop structure; this structural difference often results in higher thermostability. In addition, they are insensitive to feedback inhibition from the product cellobiose. Therefore, continuously degrading cellulases possess unique advantages and significant application potential.

[0004] Although researchers both domestically and internationally have made significant progress in improving the catalytic activity of cellulases through protein engineering, a comprehensive and systematic theoretical understanding of the persistent endoglucanase mechanism is still lacking. This makes it difficult to meet the needs of targeted design and modification of persistent endoglucanases. Therefore, this invention, based on caver and hotspot-guided enzyme modification methods, screens a library of key site mutants and performs molecular modification, providing a class of persistent endoglucanase mutants with significantly enhanced enzyme activity, offering new feasibility for the industrial biotransformation of cellulose raw materials. Summary of the Invention

[0005] The purpose of this invention is to provide an endonuclease mutant and its application, thereby solving the problem of low activity of existing endonucleases, which restricts the industrial bioconversion of cellulose raw materials.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides an endonuclease mutant, wherein the mutant is an amino acid sequence after substitution mutation at any one or more amino acid sites at positions 105, 205, and 233 of the amino acid sequence shown in SEQ ID NO.1.

[0008] Preferably, the amino acid substitution mutation at position 105 is the substitution of asparagine at position 105 of the amino acid sequence with any one of alanine, glutamic acid, threonine, or histidine.

[0009] The amino acid substitution mutation at position 205 is the substitution of threonine at position 205 of the amino acid sequence with any one of arginine, lysine, serine or phenylalanine.

[0010] The amino acid substitution mutation at position 233 is the substitution of aspartic acid at position 233 of the amino acid sequence with any one of alanine, glutamic acid, asparagine, or leucine.

[0011] Preferably, the mutant is an amino acid sequence in which asparagine at position 105 is replaced by histidine, threonine at position 205 is replaced by serine, and aspartic acid at position 233 is replaced by leucine.

[0012] Preferably, the amino acid sequence of the mutant is shown in any one of SEQ ID NO.3 to SEQ ID NO.9.

[0013] The present invention provides a nucleotide sequence encoding the mutant.

[0014] The present invention provides an expression vector containing the said nucleic acid sequence.

[0015] The present invention provides a host cell containing the expression vector.

[0016] The present invention also provides the application of the mutant, the nucleotide sequence, the expression vector, or the host cell in cellulose biodegradation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention employs NDT codon and single-point mutagenesis techniques, combining Caver tunneling guidance and Hotspot precise prediction technology, to discover the influence of key structures (tunnels) in enzyme structures on enzyme catalytic performance, resulting in a class of sustained endonucleases with significantly enhanced enzyme activity. The sustained endonucleases described in this invention exhibit high catalytic activity, providing new feasibility for the industrial bioconversion of cellulose raw materials. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The electrophoresis results for each mutant in Example 2 are shown below. M represents the protein marker, and lanes 1-26 represent the purified enzymes of mutants H36A, W40A, A65A, Y67A, I103A, L104A, N105A, G107A, N142A, G143A, T177A, S179A, Q180A, T205A, H206A, G207A, Q208A, F209A, L210A, D233A, A234A, S235A, G236A, N263A, K267A, and E269A, respectively.

[0021] Figure 2 The relative hydrolytic activity of alanine mutations at 24 key amino acid sites in Example 3 on the soluble substrate CMC;

[0022] Figure 3 The relative hydrolytic activity of alanine mutations at 24 key amino acid sites in Example 3 on the insoluble substrate Avicel;

[0023] Figure 4 The relative hydrolytic activity of the mutant in Example 3 against CMC and Avicel;

[0024] Figure 5 The relative hydrolytic activities of each combined mutant in Example 3 to CMC(A) and Avicel(B) substrates;

[0025] Figure 6 The optimal reaction temperature (left) and temperature stability (right) of the mutant and the original enzyme in Example 4 are shown in the figure.

[0026] Figure 7 The optimal pH (left) and pH stability (right) results for the mutant and original enzyme in Example 4 are shown.

[0027] Figure 8 The persistence of the original enzyme EG5C-1 and the optimal mutant in Example 5;

[0028] Figure 9The figures show the hydrolysis of the original enzyme EG5C-1 and the optimal mutant in Example 6. G1 represents glucose, G2 represents cellobiose, G3 represents cellotriose, G4 represents cellotetraose, and G5 represents cellopentaose. In the left middle figure, M represents the product standard sample; wells 1 and 8 are controls; wells 2, 3, and 4 represent the hydrolysis products of EG5C-1 on CMC, Avicel, and PASC, respectively; and wells 5, 6, and 7 represent the hydrolysis products of N105H / T205S / D233L on CMC, Avicel, and PASC, respectively. In the right middle figure, M represents the product standard sample; wells 1, 2, 3, and 4 represent the hydrolysis products of EG5C-1 on cellobiose, cellotriose, cellotetraose, and cellopentaose, respectively; and wells 5, 6, 7, and 8 represent the hydrolysis products of N105H / T205S / D233L on cellobiose, cellotriose, cellotetraose, and cellopentaose, respectively.

[0029] Figure 10 The hydrolytic performance of the original enzyme EG5C-1 and the optimal mutant on filter paper in Example 7 is shown. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Screening of key amino acid residues guided by Caver and Hotspot

[0032] The crystal structure of wild-type EG5C-1 (PDB ID: 3PZT) was predicted using a homology model of the BsCel5A catalytic module, and cellohexasaccharide was docked into the substrate channel using Autodock software. The homology model was analyzed using CaverDock 1.1 software, and tunnels Tunnel1, Tunnel2, and Tunnel3 were obtained with catalytic active sites E140 and E228 as the center points. Subsequently, the areas surrounding each tunnel were screened based on their center points. The amino acid residues are H36, W40, R63, A65, Y67, H102, I103, L104, N105, G107, N139, E140, N142, G143, T177, W178, S179, Q180, H200, F201, W202, T205, H206, G207, Q208, F209, L210, E228, D233, A234, S235, G236, W262, N263, K267, and E269.

[0033] The obtained homology model structure was input into Hotspot to predict hotspot residues in enzyme-substrate interactions. Based on the mutation score, amino acid sites with moderate and high scores were screened, namely H36, W40, A65, Y67, I103, L104, N105, G107, N142, G143, T177, S179, Q180, T205, H206, G207, Q208, F209, L210, D233, A234, S235, G236, N263, K267, and E269.

[0034] Finally, the intersection of the two methods was selected as the final key amino acids, namely H36, W40, A65, Y67, I103, L104, N105, G107, N142, G143, T177, S179, Q180, T205, H206, G207, Q208, F209, L210, D233, A234, S235, G236, N263, K267, and E269.

[0035] Example 2 Construction of a persistent endoglucanase mutant

[0036] The mutation sites obtained in Example 1 were subjected to alanine mutation and site-directed mutagenesis (NDT codon). Using the method disclosed by BinWu (Biotechnology for Biofuels, 2018, 11:20), a recombinant plasmid containing SEQ ID NO.2 (original enzyme nucleic acid sequence) was constructed. Using the sequence shown in SEQ ID NO.2 as a template, and referring to the Vazyme biological products and operation manual, the site-directed mutagenesis sequence was amplified from the whole plasmid using the corresponding mutation primers designed below. The primers used are shown in Table 1 and SEQ ID NO.10 to SEQ ID NO.99.

[0037] Table 1. Corresponding mutation primers for mutation sites

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] The codons corresponding to different amino acids for NDT are as follows: TTT (Phe), CTG (Leu), ATT (Lle), GTT (Val), TAT (Tyr), CAT (His), AAT (Asn), GAT (Asp), TGT (Cys), CGT (Arg), TCA (Ser), GGC (Gly); AHN is the degenerate codon that pairs with NDT; in the sequence listing, NDT and AHN are both replaced by NNN.

[0046] The PCR reaction system is as follows: Primer-F 1 μL, Primer-R 1 μL, Template 1 μL, ddH2O 9.5 μL, 2×PhantaMax MasterMix 12.5 μL

[0047] The PCR program was set as follows: 95℃, 3 min; 95℃, 15 s; 60℃, 15 s; 72℃, 8 min; 30 cycles; 72℃, 10 min; 4℃, Hold.

[0048] After the full plasmid amplification was completed, 2 μL of the PCR product was taken for nucleic acid electrophoresis verification. After verification, the initial template was degraded using DpnI digestive enzyme.

[0049] The digestion system was as follows: PCR product 1 μL, DpnI enzyme 1 μL, 10×QuickCut Buffer 2 μL.

[0050] The digestion procedure is as follows: 37℃, 30 min.

[0051] After digestion, the PCR products were transformed into competent E. coli BL21(DE3) cells using the heat shock method and plated on LB agar plates containing 100 μg / mL kanamycin sulfate, and incubated at 37°C for 15 h. The mutation results were verified by sequencing (performed by Anhui General Biotechnology Co., Ltd.), and the corresponding mutants were obtained.

[0052] The constructed mutants and the original enzyme EG5C-1 were inoculated into 50 mL of LB liquid medium, and kanamycin sulfate was added to a final concentration of 100 μg / mL. The culture was incubated overnight at 37°C and 180 rpm. 2% of the overnight culture was inoculated into 50 mL of fresh LB liquid medium and incubated at 37°C and 180 rpm until the OD600 reached 0.8. IPTG (final concentration 0.1 mmol / L) was then added, and expression was induced at 25°C for 24 h. The induced fermentation broth was centrifuged at 12000 rpm for 20 min, the supernatant was discarded, and the cells were resuspended in 50 mM Na2HPO4-KH2PO4 (pH 6.0) buffer. The supernatant was then sonicated and filtered through a 0.22 μm filter to obtain the crude enzyme solution. The target protein was then isolated and purified using a nickel column (GE Healthcare, Fairfield, USA) to obtain the mutants. Electrophoresis and amino acid sequencing were performed on each mutant. The electrophoresis results are as follows: Figure 1 As shown, the induced mutants all exhibit a distinct band at 33.4 kDa, and the sequencing results indicate that the mutant sequence is correct, demonstrating that the persistent endoglucanase mutant was successfully induced to express.

[0053] Example 3: Screening of the optimal mutant of persistent endonuclease

[0054] The mutant obtained in Example 2 was used as a substrate with sodium carboxymethyl cellulose (CMC) and microcrystalline cellulose Avicel to determine the changes in enzyme activity. The relative hydrolytic activity of alanine mutations at 24 key amino acid sites (sites A65 and A234 are alanine and were not mutated, therefore their activity was not measured) against the soluble substrate CMC was as follows: Figure 2 As shown, the relative hydrolytic activity to the insoluble substrate Avicel is as follows: Figure 3 As shown.

[0055] Depend on Figure 2 and Figure 3 It is evident that alanine mutations at the vast majority of sites result in loss of activity, indicating the importance of this site for enzyme catalytic activity. A few sites show significantly enhanced activity after alanine mutations, while a few others show no significant change in activity after mutation.

[0056] Subsequently, sites that were not mutated, as well as sites with activity increases or decreases greater than 20%, were selected for further study. These sites were H36, W40, A65, Y67, I103, L104, N105, G107, N142, Q180, T205, H206, L210, D233, A234, G236, K267, and E269. Mutations were performed on these 18 sites using the NDT codon. The primers used for mutation were the same as in Example 2. The relative hydrolytic activities of the mutants against CMC and Avicel are as follows: Figure 4 As shown (since sites H36, W40, I103, L104, N142, Q180, H206, A234, G236, K267, and E269 almost completely lose their activity after NDT codon saturation mutation, therefore...) Figure 4 (Not shown in the image).

[0057] Depend on Figure 4 It was found that the relative activities of mutants at sites A65, Y67, G107, and L210 were not significantly increased compared to the original enzyme, while the relative activities of mutants N105, T205S, and D233L were increased compared to the original enzyme. Among them, N105H (amino acid sequence as shown in SEQ ID NO.3), T205S (amino acid sequence as shown in SEQ ID NO.4), and D233L (amino acid sequence as shown in SEQ ID NO.5) showed the highest activities. Their activities against the soluble substrate CMC were increased by an average of 21%, 23%, and 96%, respectively, and their activities against the insoluble substrate Avicel were increased by 25%, 21%, and 30%, respectively.

[0058] Finally, based on the better mutants in each mutant library, combined mutants were designed: N105H / T205S (amino acid sequence as shown in SEQ ID NO. 6), N105H / D233L (amino acid sequence as shown in SEQ ID NO. 7), T205S / D233L (amino acid sequence as shown in SEQ ID NO. 8), and N105H / T205S / D233L (amino acid sequence as shown in SEQ ID NO. 9). Their relative hydrolytic activities against CMC and Avicel substrates were determined, and the results are as follows: Figure 5 As shown. By Figure 5 It can be seen that the activities of the combined mutants are improved to varying degrees compared with the original enzyme. Among them, the combined mutant N105H / T205 / D233L has a 2.1-fold increase in hydrolytic activity for CMC substrates and a 1.25-fold increase in hydrolytic activity for Avicel substrates compared with the original enzyme.

[0059] Example 4 Enzymatic properties of persistent endoglucanase mutants

[0060] Methods for determining the hydrolytic activity of persistent endoglucanase mutants:

[0061] Enzyme activity unit definition: One enzyme activity unit is defined as the amount of enzyme required to produce 1 mmol of reducing sugar from the substrate per minute under conditions of 60°C and pH 6.0.

[0062] (1) Endo-glucanase: Accurately weigh 1g of sodium carboxymethyl cellulose (CMC-Na) and dissolve it in 100mL of Na2HPO4-KH2PO4 buffer (50mM, pH 6.0). Stir and mix well. Accurately pipette 1.5mL into a test tube as the enzyme reaction substrate. After preheating at 60℃ for 5min, add 0.5mL of appropriately diluted protease solution. Place the tube in a 60℃ water bath and shake for 10min. Add 3mL of DNS reagent and react in a boiling water bath for 5min. Then quickly cool to room temperature. Use the inactivated enzyme reaction solution as a control to measure the absorbance value at a wavelength of 540nm.

[0063] (2) Exo-glucanase: Accurately weigh 10g of microcrystalline cellulose (Avicel) and dissolve it in 100mL of Na2HPO4-KH2PO4 buffer (50mM, pH 6.0). Stir and mix well. Accurately pipette 1.5mL into a test tube as the enzyme reaction substrate. After preheating at 60℃ for 5min, add 0.5mL of appropriately diluted protease solution. Place the tube in a 60℃ water bath and shake for 30min. Centrifuge and collect the supernatant. Add 3mL of DNS reagent. After boiling in a water bath for 5min, quickly cool to room temperature. Use the inactivated enzyme reaction solution as a control to measure the absorbance value at a wavelength of 540nm.

[0064] (3) Filter paper enzyme activity (FPase): Accurately weigh 0.5g of dried filter paper (FP), add it to 1.5mL of Na2HPO4-KH2PO4 buffer (50mM, pH 6.0), preheat at 60℃ for 5min, add 0.5mL of appropriately diluted protease solution, place in a 60℃ water bath shaker for 30min, centrifuge and collect the supernatant, add 3mL of DNS reagent, react in a boiling water bath for 5min, and then quickly cool to room temperature. Use the inactivated enzyme reaction solution as a control to measure the absorbance value at a wavelength of 540nm.

[0065] Enzyme activity X = [reducing sugar content / 180 / 10(30)] / n

[0066] Where X is the specific enzyme activity, U / mg; 180 indicates the conversion of reducing sugar from milligrams to micromoles; 10(30) is the reaction time; and n is the content of the reacting protein, mg.

[0067] 1. Optimal reaction temperature and temperature stability

[0068] The original enzyme (SEQ ID NO.2) and the N105H / T205S / D233L mutant enzyme solution obtained in Example 3 were diluted to a certain concentration, and 500 μL of each solution was added to a test tube containing 1.5 mL of CMC substrate. The reactions were carried out at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 10 min, respectively. After the reaction, 3 mL of DNS solution was added and the mixture was boiled for 5 min. The enzyme activity was then measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated sequentially. A curve showing the change of enzyme activity with temperature was plotted.

[0069] To determine the temperature stability of the N105H / T205S / D233L mutant and the original enzyme, diluted enzyme solutions were incubated in water baths at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 2 hours, respectively. The remaining enzyme activity was then measured using the same method as for determining endonuclease activity. With the highest enzyme activity defined as 100%, the relative enzyme activity was calculated sequentially, and activity change curves under different incubation conditions were plotted.

[0070] The results are as follows Figure 6 As shown, where, Figure 6 The left side shows the optimal reaction temperature results for the mutant and the original enzyme, while the right side shows the temperature stability results for the mutant and the original enzyme. It can be seen that the optimal reaction temperature for both the continuous endoglucanase EG5C-1 and the mutant N105H / T205S / D233L is 60℃. When the temperature is between 30℃ and 60℃, the enzyme activity of both EG5C-1 and the mutant gradually increases. When the temperature is above 60℃, the enzyme activity of both EG5C-1 and the mutant decreases significantly. After incubation at temperatures below 50℃ for 2 hours, the continuous endoglucanase EG5C-1 and its mutant N105H / T205S / D233L still retained more than 80% of their enzyme activity, indicating that the mutant enzyme achieved a balance between enzyme activity and temperature stability, and the increase in enzyme activity did not lead to a decrease in temperature stability.

[0071] 2. Optimal pH and pH stability

[0072] Prepare buffers and substrates with different pH values: citric acid-sodium citrate (pH 3.0–6.0), Na2HPO4-KH2PO4 (pH 6.0–8.0), and glycine-sodium hydroxide (pH 8.0–9.0), and prepare 1% CMC substrate reaction solutions under different pH conditions.

[0073] The original enzyme and the N105H / T205S / D233L mutant enzyme solution obtained in Example 3 were diluted to a certain concentration, and 500 μL of each solution was added to a CMC substrate reaction solution containing 1.5 mL of buffers with different pH values. The reaction was carried out at 60 °C for 10 min. After the reaction, 3 mL of DNS solution was added and the mixture was boiled for 5 min. The enzyme activity was then measured. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated sequentially. A curve showing the change in enzyme activity with substrate pH was plotted.

[0074] The diluted enzyme solution was diluted with buffers of different pH values ​​and incubated on ice at 4°C for 2 hours. Then, the enzyme activity was measured using the same method as for determining endonuclease activity. The highest enzyme activity was taken as 100%, and relative enzyme activities were calculated sequentially to plot the enzyme activity change curves under different incubation conditions.

[0075] The results are as follows Figure 7 As shown, where, Figure 7 The left side shows the optimal pH measurement results for the mutant and the original enzyme, while the right side shows the pH stability measurement results for the mutant and the original enzyme. The optimal reaction pH for both the continuous endoglucanase EG5C-1 and the mutant N105H / T205S / D233L is pH 6.0. Both EG5C-1 and its mutant exhibit the highest hydrolytic activity at pH 6.0, with activity decreasing as pH increases or decreases. The stability of both EG5C-1 and the mutant N105H / T205S / D233L is relatively stable between pH 5.0 and 9.0, retaining over 70% of their enzyme activity after 2 hours of incubation under various conditions. However, the stability of both EG5C-1 and its mutant decreases between pH 3.0 and 5.0.

[0076] Example 5: Persistent ability of the optimal mutant of persistent endoglucanase

[0077] The persistence of persistent endoglucanase was determined by measuring the ratio of soluble to insoluble reduced ends produced from the degradation of regenerated amorphous cellulose (PASC). The assay procedure is as follows:

[0078] (1) Dilute the N105H / T205S / D233L mutant enzyme solution obtained in Example 3 to a certain concentration (to about 0.007 mg / mL) and add 500 μL to a test tube containing 1.5 mL of 1% PASC substrate (w / v) and mix well.

[0079] (2) After reacting at 60℃ for 30 min, 60 min, 120 min and 180 min, the test tubes were taken out and immediately boiled in boiling water for 5 min to terminate the reaction;

[0080] (3) Centrifuge the reactants at 12,000 rpm for 10 min;

[0081] (4) Wash the precipitate repeatedly with Na2HPO4-KH2PO4 buffer and centrifuge at least 3 times. Then add an equal volume of buffer solution to resuspend the precipitate to obtain insoluble reducing sugar.

[0082] (5) The content of reducing sugar in the supernatant and precipitate was determined by the DNS method and the ratio of the reducing end at different reaction time periods was calculated.

[0083] The results are as follows Figure 8 As shown, when the reaction time was extended from 0.5 h to 3 h, the ratio of soluble to insoluble reducing sugars produced by the original enzyme EG5C-1 increased from 2.22 to 3.30. The ratio of soluble to insoluble reducing sugars produced by the optimal mutant N105H / T205S / D233L from the hydrolysis of PASC also increased continuously with time, from 2.69 at 0.5 h to 3.66 after 3 h. Therefore, the optimal mutant N105H / T205S / D233L not only improved the activity of both endonucleases and exonucleases but also enhanced its persistence.

[0084] Example 6: Hydrolysis products of the optimal mutant of persistent endoglucanase

[0085] The purified enzyme liquid of the N105H / T205S / D233L mutant was obtained according to Example 3. The product was hydrolyzed and analyzed using CMC, Avicel, PASC, and cellooligosaccharides (cellobiose, cellotriose, cellotetraose, and cellopentaose) as substrates. The specific steps are as follows:

[0086] (1) Take an appropriate amount of diluted purified enzyme solution and mix it with substrate CMC, Avicel and PASC in a 1:1 ratio;

[0087] (2) After the reaction solution is placed at 45℃ for 6h (CMC, Avicel, PASC) and 3h (cellulose oligosaccharide), the reaction solution is boiled to inactivate the enzyme.

[0088] (3) Centrifuge the reaction solution at 12,000 rpm for 10 min, discard the precipitate and collect the supernatant enzymatic hydrolysis product;

[0089] (4) Add pre-cooled ethanol to the hydrolysis product, let it precipitate overnight to remove impurities, and then dry it.

[0090] (5) Add appropriate amount of distilled water to dissolve the product to obtain the hydrolysis product.

[0091] Thin-layer chromatography (TLC) detection:

[0092] (1) Place the thin plate in a 120℃ oven (to remove volatile impurities and moisture and improve separation efficiency) for 1 hour to activate it and then place it in a desiccator for later use.

[0093] (2) Preparation of developing solvent (about 60 mL): Mix n-butanol: acetic acid: water = 3:2:1, add to the developing tank for about 20 min before the experiment to prevent edge effect;

[0094] (3) Spotting: The spotting volume of the standard is 1 μL (glucose, cellobiose, cellotriose, cellotetraose), and the spotting volume of each sample is 2 μL;

[0095] (4) Place the developing chamber in a fume hood and develop the layers twice at room temperature, each time for about 1.5 hours;

[0096] (5) Sample color development: Prepare color development agent (about 20 mL): Mix concentrated sulfuric acid: ethanol = 1:9 (v:v), spray the color development agent evenly on the surface of the thin plate and dry it with a hair dryer, and then put it in a 120℃ oven for color development for 5 min;

[0097] (6) Determine the hydrolysis products of EG5C-1 and each mutant based on the standard.

[0098] Experimental results are as follows Figure 9 As shown, the hydrolysis products of the sustained endonuclease EG5C-1 and the optimal mutant N105H / T205S / D233L for CMC, Avicel, and PASC substrates are mainly cellobiose and cellotriose. EG5C-1 and the mutant N105H / T205S / D233L exhibit the same hydrolysis pattern for cellooligosaccharides, hydrolyzing cellotriose, tetrasaccharides, and pentasaccharides, but not cellobiose.

[0099] Example 7 Hydrolytic performance of filter paper by the optimal mutant of persistent endonuclease

[0100] 2.5% (w / v) dried filter paper (Cytiva) was mixed with Na₂HPO₄-KH₂PO₄ (50 mM, pH 6.0), and 40 μg / mL of the sustained endonuclease EG5C-1 and its optimal mutant N105H / T205S / D233L enzyme solution were added separately. The mixture was shaken at 200 rpm and 45 °C for 24 h. A mixture without enzyme solution was used as a control to study the hydrolytic performance of the original enzyme and the mutant on the filter paper. The hydrolytic effect is as follows: Figure 10 As shown in the figure, except for the control group, the filter paper solution became turbid and the filter paper showed a fine fibrous state. Among them, the filter paper of the mutant group was almost completely hydrolyzed into filter residue after 24 hours, indicating that the mutant has higher hydrolytic performance than the original enzyme.

[0101] As can be seen from the above embodiments, the present invention provides an endonuclease mutant and its application. The obtained persistent endonuclease mutant has significantly improved enzyme activity and high catalytic activity, providing new feasibility for the industrial bioconversion of cellulose raw materials.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An endonuclease mutant, characterized in that, The mutant is any one of the following: (1) The asparagine at position 105 of the amino acid sequence shown in SEQ ID NO.1 is mutated to histidine, and the amino acid sequence of the mutant is shown in SEQ ID NO.3; (2) The amino acid sequence shown in SEQ ID NO.1 has an asparagine at position 105 mutated to histidine and a threonine at position 205 mutated to serine, and the amino acid sequence of the mutant is shown in SEQ ID NO.6; (3) The asparagine at position 105 of the amino acid sequence shown in SEQ ID NO.1 is mutated to histidine, and the aspartic acid at position 233 is mutated to leucine, and the amino acid sequence of the mutant is shown in SEQ ID NO.7; (4) The amino acid sequence shown in SEQ ID NO.1 is modified such that the asparagine at position 105 is mutated to histidine, the threonine at position 205 is mutated to serine, and the aspartic acid at position 233 is mutated to leucine, and the amino acid sequence of the mutant is shown in SEQ ID NO.

9.

2. The nucleotide encoding the mutant of claim 1.

3. An expression vector comprising the nucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3, characterized in that, The host cell is Escherichia coli.

5. The use of the mutant of claim 1, or the nucleotide of claim 2, or the expression vector of claim 3, or the host cell of claim 4 in the biodegradation of cellulose.

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