Heat-resistant persistent endoglucanase mutant with improved activity and application of heat-resistant persistent endoglucanase mutant

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to a heat-resistant persistent endoglucanase mutant with improved activity and an application of the heat-resistant persistent endoglucanase mutant. The heat-resistant persistent endoglucanase mutant with the improved activity, provided by the invention, is obtained by carrying out site-directed mutagenesis on endoglucanase DbCel5A from D. bacterium, and the specific mutation sites are K27G, V155E and A207V. According to the mutant K27G with the optimal performance, the catalytic efficiency is improved by 2.5 times, the substrate affinity is enhanced, meanwhile, the thermal stability is further improved, and a high-performance biocatalyst is provided for industrial cellulose saccharification.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, and more specifically, relates to a thermostable, persistent endoglucanase mutant with enhanced activity and its applications. Background Technology

[0002] The widespread application of natural cellulases in industry is often limited by their insufficient thermal stability. Thermophilic enzymes that maintain high catalytic activity at high temperatures are considered highly promising biocatalysts in the conversion of lignocellulose. Traditionally, cellobiases / exocellulose exoglycoses were considered the only cellulases capable of continuously degrading cellulose. Subsequent studies have found that some endoglucanases also possess the ability to continuously hydrolyze cellulose, exhibiting dual endo- and exo-catalytic functions. They can not only cleave internal glycosidic bonds within the amorphous regions of cellulose but also continuously slide along the cellulose chain, releasing soluble reducing sugars. Compared to traditional non-continuous endoglucanases, they produce higher yields of soluble oligosaccharides. This unique mechanism enables more thorough decomposition of cellulose, making them promising biocatalysts in biomass conversion. However, the number of characterized continuous endoglucanases remains limited, and very few possess the excellent thermal stability required for demanding industrial applications. This situation highlights the urgent need to discover novel continuous endoglucanases that combine higher stability and activity. Summary of the Invention

[0003] The purpose of this invention is to provide a thermostable, persistent endoglucanase mutant with enhanced activity and its applications, in order to solve the aforementioned technical problems.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a thermostable, sustained-release endoglucanase mutant with enhanced activity. The amino acid sequence of the thermostable, sustained-release endoglucanase mutant is obtained by mutation of the sequence shown in SEQ ID NO.2, and the mutants are K27G, V155E, and A207V. K27G refers to the mutation of lysine at position 27 of the N-terminus of the sequence shown in SEQ ID No.2 to glycine; V155E refers to the mutation of valine at position 155 of the N-terminus of the sequence shown in SEQ ID No.2 to glutamic acid; and A207V refers to the mutation of alanine at position 207 of the N-terminus of the sequence shown in SEQ ID No.2 to valine.

[0005] Addressing the technical bottleneck of lacking a sustained endoglucanase with both high thermal stability and high catalytic activity in the industrial cellulose saccharification process, this invention addresses the issue from two levels: enzyme discovery and protein engineering. First, based on the known thermostable template enzyme TmCel5A, DbCel5A, derived from thermophilic microorganisms, was screened as the starting enzyme through sequence homology and phylogenetic analysis. This enzyme possesses excellent thermal stability and sustained hydrolytic ability. Subsequently, through rational design combined with site-directed mutagenesis, a series of mutants, including K27G, V155E, and A207V, were constructed. Among them, the K27G single-site mutant showed the most outstanding performance, with its catalytic efficiency increasing by approximately 2.4 times compared to the wild type, and its thermal denaturation temperature (Tm) simultaneously increasing, achieving synergistic optimization of "dual enhancement of stability and activity." This mutant can react efficiently with cellulose substrates, with the main products being glucose and cellobiose, making it suitable for harsh industrial environments such as high temperature and high salt. This invention not only provides a high-performance mutant enzyme applicable to industrial applications but also establishes a complete technical path from enzyme discovery and characterization to rational modification.

[0006] The present invention also provides a nucleic acid molecule that encodes the thermostable, persistent endoglucanase mutant.

[0007] The present invention also provides a carrier comprising the aforementioned nucleic acid molecule.

[0008] Furthermore, the vector is a cloning vector or an expression vector.

[0009] The present invention also provides a recombinant cell comprising the aforementioned vector.

[0010] Furthermore, the engineered bacteria is Escherichia coli.

[0011] Furthermore, the *E. coli* is E. coli BL21(DE3).

[0012] The present invention also provides the application of the heat-resistant, persistent endoglucanase mutant in the catalytic hydrolysis of cellulose.

[0013] Furthermore, the catalytic temperature is 50°C to 60°C.

[0014] The present invention has at least the following beneficial effects: The present invention Db Cel5A exhibits excellent thermal stability and salt resistance. Its high thermal denaturation temperature allows it to maintain its activity for extended periods under high-temperature conditions, making it suitable for high-temperature cellulose hydrolysis processes. Its hydrolysis products are mainly glucose and cellobiose, which helps improve cellulose saccharification efficiency and demonstrates its advantages in one-step saccharification.

[0015] Based on this, the mutant K27G obtained through enzyme engineering significantly improved catalytic efficiency and substrate affinity while maintaining or enhancing thermal stability, thus achieving comprehensive optimization of enzyme performance.

[0016] Therefore, this invention provides a thermostable, sustained-release endoglucanase and its mutants that combine high stability and high catalytic efficiency, and establishes a widely applicable method for the discovery and engineering modification of high-performance cellulases, which has good prospects for industrial application. Attached Figure Description

[0017] Figure 1 This is a sequence mining analysis diagram of potential persistent endoglucanases, where A represents candidate enzymes and... Tm Phylogenetic relationships among Cel5A, and B is a sequence similarity network of candidate persistent endoglucanases.

[0018] Figure 2 This is an enzyme expression diagram, where A represents... Db SDS-PAGE analysis of the Cel5A purification process, B is... Db Substrate-specific detection diagram of Cel5A.

[0019] Figure 3 for Db Biochemical characteristics analysis diagram of Cel5A, where A represents... Db The optimal reaction temperature diagram for Cel5A is shown, with B representing... Db Thermal stability diagram of Cel5A, where C represents... Db The optimal reaction pH for Cel5A is shown in the diagram, where D represents... Db pH stability diagram of Cel5A, E is Db The thermal deactivation half-life of Cel5A at 55℃, 65℃, 70℃, and 75℃ is F. Db The thermal deactivation half-life of Cel5A at 55°C and 65°C.

[0020] Figure 4 for Db The graph shows the influence of chemical reagents and synergistic activity of Cel5A, where A represents the effects of metal ions, organic reagents, and surfactants on... Db The effect of NaCl concentration on Cel5A activity is shown in Figure B, where B represents the effect of NaCl concentration on Cel5A activity Db The effect of Cel5A activity is shown in the figure.

[0021] Figure 5 for Db Product analysis and hydrolysis characteristics of Cel5A, where A represents... Db The HPAEC−PAD analysis results of CMC hydrolyzed by Cel5A after 1 h, 3 h, and 24 h are shown in Figure B. Db Persistence of Cel5A during filter paper hydrolysis.

[0022] Figure 6 This is a characterization diagram of the mutant enzyme, where A represents the purified enzyme. Db SDS-PAGE analysis of Cel5A WT and its mutants, B is the catalytic activity determination of WT and mutants at 55℃ and pH 4.0. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0024] Example 1: Enzyme purification.

[0025] I. Experimental Materials.

[0026] pET-22b(+) and pET28a-CelOCE were synthesized by Sangon Biotech (Shanghai, China). Mutant primers were synthesized by J.L. Biotech (Shanghai, China). Reagents used in the experiment included the StarPrep Fast Plasmid Mini Kit (GenStar, Beijing, China), 2×Phanta Max Master Mix (Vazyme, Jiangsu, China), BCA Protein Quantitative Kit (Beyotime, Shanghai, China), sodium carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) (Aladdin, Shanghai, China), Whatman No.1 filter paper (Cytiva, Shanghai, China), peptone and yeast extract (Oxoid, UK), and other chemical reagents (Sinopharm Chemical Reagents, Beijing, China).

[0027] II. Discovery of novel enzymes and bioinformatics analysis.

[0028] To meet the current industrial demand for thermostable, persistent endoglucanases, a systematic screening and analysis of target enzyme sequences was conducted. To achieve accurate and efficient identification of thermostable, persistent endoglucanases, [the following steps were taken]. Tm Sequences with Cel5A amino acid sequence identity in the range of 50% to 70% are considered potential functional candidate enzymes. Tm The accession number for the Cel5A amino acid sequence is AAD36816.1.

[0029] This invention aims to screen for persistent endoglucanases with excellent thermostability, focusing on candidate sequences derived from thermophilic groups. Ultimately, one sequence derived from... D. bacterium GH family enzymes DbCel5A (GenBank accession number: HOP94420.1) was used as the research object, such as Figure 1 As shown in A, its phylogenetic relationship is related to the template enzyme. Tm Cel5A is the closest. Visualization of sequence similarity can be found in [link to visualization]. Figure 1 B.

[0030] three, Db Expression and purification of Cel5A.

[0031] Db After codon optimization, the Cel5A gene yielded the sequence SEQ ID No. 1, which was then inserted into the pET-22b(+) vector. Nde I and Xho At site I, the target gene is carried. Db The recombinant plasmid of Cel5A was transformed into the expression host. E. coli BL21(DE3) was successfully screened for transformants on LB agar plates containing ampicillin. A single confirmed colony was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and incubated overnight at 37°C and 220 rpm. Subsequently, the culture was amplified to 200 mL of LB medium and incubated at 37°C until OD500. 600 When the concentration reaches 0.7, add isopropyl- to a final concentration of 0.5 mM. β Protein expression was induced by β-D-thiogalactoside (IPTG) and cultured at 28°C for 7 h.

[0032] SEQ ID No.1:ATGGAAGTTTATAAACTGCCGATCTACCGTGGCATCAACATGGGTGATGCTCTGGAAGCGCCGGTGGAAGGTGGCTGGAAAGTTGTTATCAAAGATGAATATTTCAAACTGATGAAAGAAGCTGGCTTCGATCACGTTCGTATCCCGATCAAATGGAGCGCGCACACCGAAGCGAAACCGCCGTACAAAATCTCTGAAGAATTCTTCGATCGTGTTGATCACGTTATCAACGAAAGCCTGAAACAGGAACTGATTACCATCATCAACATCCACCACTACGATGAAATCATGCAGAACCCGCGTGGCGAAAAAGAAAAATTCCTGAGCATCTGGAAACAGATCAGCGAACGTTACAAAGATTACCCGGAAACCCTGTTCTTCGAAATCCTGAACGAACCGAACGGCAACCTGACCCCGGATATCTGGAACGAATTCCTGGCGGAAGCGCTGAAAGTTATCCGTGTTACCAACCCGGATCGTGTTGTTCTGGTTGGTACCGCTGAATGGGGTGGCATCAGCAGCATCTCTAAACTGAAAATCCCGAAAGAAGAGAAAAACATCCTGGTTACCGTTCACTACTACAACCCGTTCTATTTCACCCACCAGGGTGCTGAATGGGCTAGCGGTAGCGAACAGTGGCTGGGTACCAAATGGCACGGCTCCTGGGCGGAAAAACAGCAGGTTATCTCCGATTTCAACATCGCGGAAGAATGGAGCAAAGAAAACCGTCGTCCGATCCACATCGGCGAATTCGGTGCGTACAGCAAAGCGGATATGGAATCCCGTGTGCGTTGGACCAGCTTCGTTGCGCGCGAAGCGGAACGTCGTGGCTGGCCGTGGACCTACTGGGAATTCTGCAGCGGTTTCGGCGTTTACGATCCGGTTAAAAACGAATGGCGTAAAGAACTGCTGGAAGCGCTGATCCCGATCGATAAA。

[0033] After induction, cells were harvested by centrifugation at 8000 rpm for 10 min at 4°C and resuspended in 25 mL of lysis buffer (50 mM citrate-phosphate buffer, CPBS, pH 7.4). Cell disruption was performed using an sonicator under ice-water bath conditions (pulse on for 1 s, off for 2 s, total duration 25 min). Cell debris was removed by centrifugation at 12000 rpm for 30 min at 4°C to obtain crude enzyme solution.

[0034] The crude enzyme solution was loaded onto a Ni-NTA Beads 6FF column (Smart-Lifesciences Biotech, China) for His-tag affinity purification. Non-specifically bound proteins were removed using elution buffer (50 mM PBS, 50 mM imidazole, pH 7.4), followed by elution of the target protein using elution buffer (50 mM PBS, 300 mM imidazole, pH 7.4). The purified protein was analyzed by 12.5% ​​SDS-PAGE, and its concentration was determined using a BCA protein quantification kit. SDS-PAGE analysis of the purified protein showed a clear single band with a molecular weight of approximately 35 kDa. Figure 2 As shown in A, it indicates Db Cel5A can be expressed in a soluble and efficient manner.

[0035] Example 2: Db Enzymatic properties of Cel5A.

[0036] I. Substrate-specific detection.

[0037] Determining using the DNS method Db Cel5A exhibits enzymatic activity against MCC, filter paper, phosphate-expanded cellulose (PASC), β-D-glucan, and glucomannan. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under specified conditions.

[0038] like Figure 2 As shown in Figure B, this enzyme exhibits high catalytic activity towards soluble substrates (including CMC and glucomannan), demonstrating its typical endoglucanase characteristics and potential ability to degrade hemicellulose (glucomannan). In contrast, its catalytic activity towards insoluble substrates such as MCC and filter paper is significantly lower. This substrate-specific characteristic indicates strong activity towards soluble polysaccharide substrates.

[0039] II. Reaction temperature detection.

[0040] Using CMC as a substrate, the effects of temperature, pH, metal ions, and organic reagents on the surface quality of the substrate were investigated. DbThe effect of Cel5A activity was assessed. Temperature characteristics were determined by reacting the enzyme at 10°C intervals for 20 min within the range of 30°C to 80°C, followed by enzyme activity measurement to determine the optimal temperature. Thermal stability was evaluated by incubating the enzyme at the corresponding temperature for 2 h, with untreated enzymes serving as a control. The effect of pH on enzyme activity was determined within the pH range of 3 to 10 using citrate buffer (pH 3.0–6.0), PBS buffer (pH 6.0–8.0), and Gly-NaOH buffer (pH 8.0–10.0). In pH stability assays, the enzyme was incubated at 4°C for 2 h at the corresponding pH conditions, and residual activity was measured with untreated enzyme activity as 100%. The half-life at the optimal temperature was determined by incubating the enzyme and recording the time required for activity to decrease to 50%. Differential scanning fluorescence (DSF) was used to determine the protein's thermal denaturation temperature (Tm) to assess thermal stability.

[0041] like Figure 3 As shown in A, Db The activity changes of Cel5A within the temperature range of 30℃ to 80℃ indicate that it exhibits the highest enzyme activity at 55℃, with the activity initially increasing and then decreasing with increasing temperature. This enzyme has a wide reaction temperature range, maintaining over 80% of its high catalytic activity for 20 minutes at temperatures between 45℃ and 70℃. However, at 80℃, the enzyme activity decreases significantly, which may be due to catalytic structural instability caused by high temperature. Db Cel5A also exhibits strong thermal stability. Figure 3 B showed that after incubation at 45℃~80℃ for 2 hours, the remaining enzyme activity remained above 70% even below 75℃.

[0042] Compared to temperature changes, Db The catalytic efficiency of Cel5A is more sensitive to pH fluctuations. Figure 3 The C-values ​​show that the optimal reaction pH for this enzyme is 4.0, and its catalytic activity depends on a weakly acidic environment. Under strongly acidic (pH < 3) and alkaline (pH > 8) conditions, enzyme activity is significantly lost; however, in the pH range of 3.5–5.0, the relative enzyme activity remains above 70%. With increasing pH to 6.0 and 8.0, the enzyme activity drops sharply to approximately 50% and 20%, respectively. However, compared to its sensitivity to pH fluctuations, Db Cel5A exhibits excellent structural stability over a wide pH range. Figure 3 The results showed that even after incubation in neutral and alkaline buffers for 2 hours, the residual enzyme activity decreased very little. This indicates that... Db Cel5A is an enzyme with slightly acidic catalytic activity but good structural stability.

[0043] because DbCel5A showed strong heat resistance in preliminary experiments, and this invention further investigated its half-life at the optimal temperature and higher temperatures. Figure 3 The results showed that at 70℃ and 75℃, enzyme activity was halved at approximately 30h and 50h, respectively; however, after incubation at 55℃ and 65℃ for 60h, enzyme activity did not decrease significantly and even slightly increased before the final decrease. Further extension of incubation time at 55℃ and 65℃ revealed that enzyme activity began to decrease after 9 days. Figure 3 The F-type activity eventually decreased to approximately 50% of its initial activity on days 15 and 18, respectively. These results indicate... Db Cel5A exhibits excellent structural stability.

[0044] III. Enzyme activity analysis.

[0045] To evaluate the effects of metal ions and chemical reagents, 5 mM and 10 mM of K⁺, Na⁺, Ca²⁺, Ni²⁺, Cu²⁺, Fe²⁺, Fe³⁺, and Mn²⁺, as well as different concentrations of EDTA, SDS, methanol, ethanol, urea, PMSF, DMSO, Triton 100, Tween-20, and Tween-80 were added to the reaction system under optimal conditions. After 20 min of reaction, enzyme activity was expressed as a control (100%) without added reagents.

[0046] like Figure 4 As shown in Figure A, among the tested metal ions, Mn²⁺ had the most significant promoting effect on enzyme activity, with 5 mM Mn²⁺ increasing the enzyme activity to 138.0% of the initial level. In contrast, the other metal ions had a smaller effect on enzyme activity, indicating that... Db Cel5A exhibits strong metal resistance.

[0047] Compared to metal ions, organic reagents and surfactants are more effective at inhibiting the growth of metal ions. Db The effect of Cel5A was more significant. Except for urea, all other chemical reagents had a slight inhibitory effect, while 1% urea increased the catalytic activity to 130.0% of the control group (no chemical additives). Among all tested factors, the anionic surfactant SDS had the most significant effect. Db Cel5A exhibits the strongest inhibitory effect. This invention evaluated... Db Salt tolerance of Cel5A under high salt conditions, the results are as follows: Figure 4 As shown in B. The results indicate that under 0.75 M NaCl conditions, Db The catalytic activity of Cel5A is almost the same as under salt-free conditions; under high-salt conditions, Db Cel5A retained approximately 86% and 73% of its activity in 3M and 4M NaCl, respectively, demonstrating strong salt tolerance. This indicates its potential application value as a biocatalyst in high-salt environments.

[0048] Example 3: Identification of hydrolysis products.

[0049] Take 500 μL of a solution with a concentration of 50 ug / ml. Db Cel5A enzyme solution was incubated with 1.5 mL of 1% CMC solution at 55 °C. The composition of cellulosic oligosaccharides in the reaction product was then analyzed by high performance anion exchange chromatography-pulse amperometric detection (HPAEC–PAD) and high performance liquid chromatography (HPLC).

[0050] HPAEC–PAD analysis: Separation was performed using a CarboPac™ PA1 column (4×250 mm, DIONEX) at 30℃, a flow rate of 0.25 mL / min, and an injection volume of 25 μL. The mobile phase consisted of phase A (0.2 mol / L NaOH) and phase B (1 mol / L NaAc dissolved in 0.2 mol / L NaOH), with a gradient elution program as follows: Phase B 0–10 min, 0%–5%; 10–25 min, 5%–10%; 25–30 min, 10%–100%; 30–36 min, 100%; 36–40 min, 100%–0%; 40–45 min, 0%.

[0051] HPLC analysis: Analysis was performed on an UltiMate 3000 system equipped with a Luna® 5 μm NH2 column (250 × 4.6 mm, Phenomenex) and a refractive index detector (RID). The column temperature was maintained at 45 °C, the flow rate was 0.5 mL / min, the mobile phase was 70% acetonitrile, and the injection volume was 25 μL.

[0052] Figure 5 A shows Db HPAEC–PAD analysis results of Cel5A hydrolysis products. The results show that... Db Cel5A mainly produces small oligosaccharides (glucose G1, cellobiose G2, and cellotriose G3) during the hydrolysis of CMC. As the reaction time was extended to 24 hours, the contents of glucose and cellobiose increased significantly, while the contents of cellotriose decreased significantly, indicating that most of the G3 molecules were further hydrolyzed into G1 and G2. Db Cel5A hydrolyzes cellulose substrates more thoroughly, with G1 and G2 as the main products. Db Cel5A exhibits a unique catalytic property, generating a wider range of hydrolysis products, demonstrating the potential for a single enzyme to achieve complete hydrolysis of cellulose in one step.

[0053] Example 4: Db Determination of the sustained hydrolysis capacity of Cel5A.

[0054] The sustained hydrolytic capacity of this endoglucanase was evaluated by measuring the distribution of soluble and insoluble reducing sugars generated during quantitative filter paper hydrolysis. Db After Cel5A was incubated with filter paper, the reaction system was separated into a supernatant and residual solids. Soluble reducing sugars in the supernatant were quantitatively analyzed using the DNS method, with D-glucose as the standard. The insoluble fraction was treated with 6M guanidine hydrochloride, followed by washing three times with 50mM CPBS buffer (pH 7.4) and drying. Then, the insoluble reducing sugars were determined using freshly prepared BCA reagent at 75°C for 30 min, again using D-glucose as the standard. The persistence index was defined as the ratio of soluble to insoluble reducing sugar content. A heat-inactivated enzyme was used as a negative control.

[0055] The persistence of endoglucanases with dual endo / exoglycosidic activities can be quantified by the soluble / insoluble reducing sugar ratio (S / I) produced by filter paper hydrolysis. An S / I ratio between 3 and 8 indicates that the enzyme exhibits persistent activity. Figure 5 As shown in B, Db After Cel5A hydrolyzed Whatman filter paper for 8 hours, the S / I value increased to 3.8, reflecting its significant and sustained hydrolysis ability. Combined with the analysis results of the hydrolysis products, this confirms... Db Cel5A functions as a class of persistent endoglucanases.

[0056] Example 5: Db Construction of Cel5A mutant.

[0057] right Db Cel5A undergoes site-directed mutagenesis. Db The Cel5A amino acid sequence is shown in SEQ ID No. 2.

[0058] SEQ ID No.2: MEVYKLPIYRGINMGDALEAPVEGGWKVVIKDEYFKLMKEAGFDHVRIPIKWSAHTEAKPPYKISEEFFDRVDHVINESLKQELITIINIHHYDEIMQNPRGEKEKFLSIWKQISERYKDYPETLFFEILNEPNGNLTPDIWNEFLAEALKVIR VTNPDRVVLVGTAEWGGISSISKLKIPKEEKNILVTVHYYNPFYFTHQGAEWASGSEQWLGTKWHGSWAEKQQVISDFNIAEEWSKENRRPIHIGEFGAYSKADMESRVRWTSFVAREAERRGWPWTYWEFCSGFGVYDPVKNEWRKELLEALIPIDK.

[0059] Mutation-specific primers were designed using the Vazyme online tool. PCR amplification was performed according to the instructions of the 2×Phanta Max MasterMix kit. The amplified products were treated with Takara Quickcut™ DpnI at 37°C to remove the wild-type (WT) template, and then transformed into *E. coli* DH5α competent cells. The correctness of the target point mutation was confirmed by DNA sequencing. The corresponding variants were successfully constructed, expressed, and purified, such as... Figure 6 As shown in A.

[0060] Y9K indicates that the tyrosine at position 9 of SEQ ID No. 2 has been mutated to lysine.

[0061] D16N indicates that the aspartic acid at position 16 of SEQ ID No. 2 has been mutated to asparagine.

[0062] K27G indicates that the lysine at position 27 of SEQ ID No. 2 has been mutated to glycine.

[0063] M38I indicates that the methionine at position 38 of the sequence SEQ ID No. 2 has been mutated to isoleucine.

[0064] H74W indicates that the histidine at position 74 of SEQ ID No. 2 is mutated to tryptophan.

[0065] V155E indicates that the valine at position 155 of SEQ ID No. 2 is mutated to glutamic acid.

[0066] A207V indicates that the alanine at position 207 of the sequence in SEQ ID No. 2 has been mutated to valine.

[0067] W222E indicates that the tryptophan at position 222 of the sequence in SEQ ID No. 2 has been mutated to glutamic acid.

[0068] W278I indicates that the tryptophan at position 278 of the sequence in SEQ ID No. 2 has been mutated to isoleucine.

[0069] C286A indicates that the cysteine ​​at position 286 of the sequence in SEQ ID No. 2 has been mutated to alanine.

[0070] Under optimal reaction conditions (55℃, pH 4.0), 500 μL of 50 μg / ml enzyme solution was reacted with 1.5 mL of CMC substrate, and the enzyme activity of WT and mutant was measured after 20 min.

[0071] Under optimal reaction conditions, substrate solutions with concentrations of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.5, 15.0, 17.5, and 20.0 mg / mL were prepared by dissolving CMC in 50 mM, pH 4.0 citrate buffer. The values ​​of Km, Kcat, and Vmax were calculated by fitting Michaelis-Menten curves, and the results are shown in Table 1.

[0072] Among these variants, the K27G, V155E, and A207V mutants showed significantly enhanced catalytic activity compared to WT, with K27G exhibiting the most significant 2.5-fold enhancement. Figure 6 Furthermore, the K27G mutation increased the enzyme's affinity for CMC substrates, decreasing Km from 13.42 mg / mL to 11.30 mg / mL and increasing the kcat / Km ratio by 2.43-fold (Table 1). Notably, the Tm of the K27G variant was also slightly increased, from 79.9 °C at WT to 81.9 °C. In conclusion, the substitution of amino acid at position 27 with glycine synergistically enhances… Db The catalytic efficiency and thermal stability of Cel5A provide valuable guidance for the engineering modification of related enzymes.

[0073] Table 1: Db Kinetic parameters and melting temperature (Tm) of Cel5A WT and K27G mutants It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0074] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0075] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A thermostable, persistent endoglucanase mutant with enhanced activity, characterized in that, The thermostable, persistent endoglucanase mutant was obtained by mutating the DbCel5A enzyme with the amino acid sequence shown in SEQ ID NO.

2. The mutants are K27G, V155E, and A207V. K27G refers to the mutation of lysine at position 27 of the N-terminus of the sequence shown in SEQ ID NO.2 to glycine. V155E refers to the mutation of valine at position 155 of the N-terminus of the sequence shown in SEQ ID NO.2 to glutamic acid. A207V refers to the mutation of alanine at position 207 of the N-terminus of the sequence shown in SEQ ID NO.2 to valine.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the thermostable, persistent endoglucanase mutant of claim 1.

3. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. The carrier according to claim 3, characterized in that, The vector is a cloning vector or an expression vector.

5. A recombinant cell, characterized in that, The recombinant cells comprise the vector as described in claim 3.

6. The engineered bacteria according to claim 5, characterized in that, The engineered bacteria is Escherichia coli.

7. The engineered bacteria according to claim 6, characterized in that, The Escherichia coli is E. coli BL21(DE3).

8. The application of the heat-resistant, persistent endoglucanase mutant of claim 1 in the catalytic hydrolysis of cellulose.

9. The application according to claim 8, characterized in that, The catalytic temperature is 50℃~60℃.

Images