Beta-n-acetylhexosaminidase mutants with improved thermal stability and catalytic performance, screening method and application thereof

Abstract

The present application provides a beta-N-acetylhexosaminidase mutant with improved thermal stability and catalytic performance, as well as a screening method and application thereof, and relates to the field of enzyme engineering. The present application comprehensively utilizes X-ray crystal structure, AlphaFold2 and AlphaFold3 predicted structure, combined with DeltaDeltaG fold computing, sequence conservation analysis, B-factor flexible residue screening and disulfide bond engineering, screening and combining beneficial mutations to obtain a combined mutant M9. Compared with the cbHEX synergistic system, the monosaccharide yield of the M9 synergistic system is increased by 26.7 times, and the degradation rate of crystalline chitin is increased by 28%. The strategy described in the present application can significantly improve the thermal stability and catalytic performance of the target enzyme, and can provide a method reference for the rational modification of enzyme molecules.

Description

Technical Field

[0001] This invention relates to β-N-acetylglucosamine enzyme mutants with improved thermal stability and catalytic performance, as well as their screening methods and applications, belonging to the field of enzyme engineering. Background Technology

[0002] Enzymes are highly efficient and environmentally friendly biocatalysts, capable of achieving highly selective biochemical transformations under mild reaction conditions, thus holding broad application prospects in industrial catalysis, biomanufacturing, pharmaceuticals, and food. However, natural enzymes often cannot withstand the high temperatures required by industrial processes, limiting their application in practical production. Therefore, developing enzyme engineering strategies that can effectively improve enzyme stability and activity is of great significance.

[0003] Directed evolution has made significant progress in improving enzymatic properties, but this method typically relies on multiple rounds of random mutations and high-throughput screening, resulting in a large experimental workload and long cycle. In contrast, rational design can combine protein structure, energy, and evolutionary information to target specific sites, thus achieving higher design efficiency. For example, regions with high B factors in crystal structures usually reflect strong local flexibility and can serve as important targets for improving stability; at the same time, sequence conservation analysis and fold free energy change calculations can also help screen for beneficial mutations that improve both stability and catalytic function.

[0004] β-N-acetylhexosaminidase (cbHEX) can further hydrolyze N-acetylated chitosan oligosaccharide to N-acetylglucosamine (GlcNAc, NAG), and can synergistically work with exochitinase to achieve the complete conversion of chitin to GlcNAc. GlcNAc is a monosaccharide with important applications in medicine, nutrition, and cosmetics. However, the optimal temperature of cbHEX is only 50°C, significantly lower than that of the thermostable exochitinase 76Chi (optimal temperature 65°C) obtained in our laboratory. This temperature mismatch leads to a decrease in synergistic hydrolysis performance. Therefore, it is urgent to improve the thermal stability of cbHEX to meet the requirements of efficient biotransformation of chitin under high-temperature conditions. Summary of the Invention

[0005] The purpose of this invention is to provide β-N-acetylglucosamine enzyme mutants with improved thermal stability and catalytic performance, as well as their screening methods and applications, so as to improve the accuracy and success rate of enzyme engineering design and achieve synergistic optimization of the thermal stability and catalytic performance of the target enzyme.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a β-N-acetylglucosamine enzyme mutant, the mutant comprising an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO:1, and comprising substitution of at least one amino acid from the following group: A29L, T102Y, D169K, G764I, S803R, M511F, T516V, E584C, S557C.

[0008] Further, the amino acid sequence of the mutant is as shown in SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID NO:13 or SEQ ID NO:14 or SEQ ID NO:15 or SEQ ID NO:16.

[0009] The present invention also provides a gene encoding the above-mentioned β-N-acetylglucosamine enzyme mutant.

[0010] The present invention also provides a recombinant vector containing the above-mentioned genes.

[0011] The present invention also provides recombinant engineered bacteria containing the above-described recombinant vector or the above-described gene.

[0012] Preferably, the recombinant engineered bacteria is Escherichia coli; more preferably, the Escherichia coli is BL21(DE3) or TOP10.

[0013] This invention further provides a method for screening the above-mentioned β-N-acetylglucosaminease mutants, comprising:

[0014] Strategy 1: Using the X-ray crystal structure of β-N-acetylhexosaminease, the AlphaFold2 (AF2) structure, and the AlphaFold3 (AF3) structure as templates, respectively, based on the folding free energy change ΔΔG fold Wild-type residue frequency Fre WT and mutation residue frequency Fre mut Candidate mutations are screened, with a strict screening criterion of ΔΔG. fold < -4.0 kcal·mol -1 And Fre mut ≥6%, the screening condition is relaxed to -4.0≤ΔΔG fold < -3.0 kcal·mol -1 And Fre mut This represents the highest frequency at that site;

[0015] Strategy 2: Based on the B factor of each residue in the crystal structure, select highly flexible residues with B factors in the top 25% as candidate sites, and then screen those that satisfy ΔΔG. fold ≤ -2.5 kcal / mol and Fre mut Fre WT Mutations;

[0016] Disulfide bond engineering: This method uses disulfide bond prediction programs to scan protein structures, screen for residue pairs with potential bonding potential, and then combines this with disulfide bond formation energy and χ² (x-coefficient of mass). 3 Dihedral angles and local B-factors of residues were used to design cysteine ​​substitutions by selecting residue pairs with the lowest disulfide bond formation energy and located in highly flexible regions. χ 3 The dihedral angle is within ±5° of -87° or +97°.

[0017] Preferably, in strategy 1, the change in folding free energy ΔG is calculated using the Cartesian_ddg protocol in the Rosetta software. fold Before energy calculation, the wild-type structure undergoes a two-step relaxation process: first, a coordinate-constrained FastRelax is applied, followed by Cartesian FastRelax optimization using the ref2015_cart energy function to obtain the energy-minimized conformation; ΔG for each mutation. fold according to Formula calculation, where This represents the folding free energy of the mutant. This represents the folding free energy of the cbHEX wild type.

[0018] On the other hand, the present invention also provides the application of the above-mentioned β-N-acetylglucosamine enzyme mutant in the synergistic exochitinase hydrolysis of chitin and the preparation of N-acetylglucosamine.

[0019] Preferably, the amino acid sequence of the mutant is SEQ ID NO:16, and the exochitinase includes thermostable exochitinase 76Chi.

[0020] The beneficial effects of this invention are:

[0021] (1) This invention employs the protein rational design strategy SDACS, which integrates protein crystal structure and AlphaFold predicted structure. SDACS combines the advantages of experimental crystal structure and AlphaFold predicted structure. Among them, the crystal structure provides higher precision atomic coordinates and B-factor flexibility information for thermal stability design, which is beneficial for global stability screening; the AlphaFold2 and AlphaFold3 models provide conformational information different from crystal structure in the active pocket region, which can be used to identify near-active site mutations with catalytic significance. This strategy breaks through the limitation of relying on a single structure source for rational design and improves the success rate of computational design.

[0022] (2) Based on the above strategy, this invention successfully obtained the β-N-acetylhexosaminease multi-point mutant M9 with simultaneously enhanced thermal stability and catalytic activity. Compared with wild-type cbHEX, the optimal reaction temperature of M9 increased from 50℃ to 65℃. m The temperature was increased from 43.5℃ to 59.5℃, and the half-life at 50℃ was extended from 4 min to 240 min (approximately 60-fold), with the catalytic efficiency increasing to 5.4 times that of the wild type. In the chitinase synergistic degradation system, the mutant M9 constructed in this invention can synergistically interact with exochitinase 76Chi at 65℃, significantly improving the conversion of crystalline chitin to N-acetylglucosamine, indicating that this invention has good industrial application potential. Attached Figure Description

[0023] Figure 1 Phylogenetic analysis of cbHEX.

[0024] Figure 2 Multiple sequence alignment analysis of cbHEX with other N-acetylglucosamineases.

[0025] Figure 3 Optimal temperature and pH curves for cbHEX.

[0026] Figure 4 Product analysis of cbHEX hydrolyzed chitin oligosaccharides (DP1-6).

[0027] Figure 5 The half-life of cbHEX at 40℃, 50℃ and 60℃ (t 1 / 2 ).

[0028] Figure 6 Substrate-specific identification of cbHEX.

[0029] Figure 7 A schematic diagram of the cbHEX crystal structure.

[0030] Figure 8The optimal temperature profiles for cbHEX and M9.

[0031] Figure 9 Analysis of the products of chitin crystalline by synergistic hydrolysis of 76Chi with cbHEX or M9. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention. Those skilled in the art can use other conventional methods, experimental schemes, and reagents based on the technical solutions described in the present invention, and are not limited to the specific embodiments of the present invention.

[0033] Example 1: Identification of β-N-acetylglucosaminease cbHEX in compost macrotranscriptome

[0034] In this study, total RNA was extracted from compost samples using the Qiagen RNA Extraction Kit (catalog number 74104) and sequenced on the Illumina HiSeq 3000 / 4000 platform. Raw sequencing data were quality-controlled and assembled using Trinity. Open reading frames (ORFs) for each transcript were predicted using TransGeneScan. Redundant sequences were clustered using CD-HIT (Cluster Database at High Identity with Tolerance), with a sequence identity threshold of 95% and a coverage threshold of 90%. The longest transcript in each cluster was retained as the representative sequence. Transcript abundance was expressed as fragments per million alignment reads (FPKM). Functional annotations were obtained by BLASTp alignment with the NCBI Non-Redundant Protein Database (NR). Amino acid sequences were further analyzed using the PSI-BLAST tool on the NCBI server. Based on sequence similarity and conservation analysis, a putative β-N-acetylhexosaminease gene was identified. The transcriptional level of this gene was significantly higher during the mid-to-late high-temperature stage (50-60℃) of composting than during the mesophilic and mature stages, indicating that the encoded enzyme may have high catalytic activity between 50-60℃. Phylogenetic analysis using the neighbor-joining method in MEGA7 software showed that this enzyme had the highest sequence homology (85.52%) with the GH20 family β-N-acetylglucosaminease (WP_094984658.1) from *Cellvibrio mixtus*, while exhibiting significant evolutionary differences from members of the GH3 and GH84 families. Figure 1 Subsequently, multiple sequence alignment was performed using Clustal Omega to compare it with GH20 β-N-acetylglucosamineases from several different microbial sources. The results showed that it had the highest structural similarity (41.33%) to its homologous protein from *Serratia marcescens* (PDB ID: 1QBA). The alignment also revealed that the enzyme possesses the conserved HXGXDE motif characteristic of the GH20 family, particularly the strictly conserved Asp514 and Glu515 key catalytic residues. Figure 2 ).

[0035] In summary, this enzyme is a GH20 β-N-acetylglucosaminease, named cbHEX, with its amino acid sequence shown in SEQ ID NO:1 and its gene sequence shown in SEQ ID NO:2. The enzyme contains 832 amino acids. Its theoretical molecular weight is 91.0 kDa, and its predicted isoelectric point (pI) is 5.9.

[0036] Example 2: Heterologous expression and property determination of cbHEX

[0037] The gene encoding cbHEX was codon-optimized and cloned into the pCold-TF vector (containing a 6×His tag and TEV restriction site at the N-terminus); the mutant was constructed into the pCold II vector. The plasmid was transformed into *E. coli* BL21(DE3) competent cells and cultured at 37°C in LB medium containing 50 mg / L ampicillin until OD500. 600 ≈0.6, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction, and expression was induced at 16℃ for 18 hours. The induced bacterial cells were collected by centrifugation and homogenized under high pressure. After adding 1% (w / w) TEV protease, the cells were purified by immobilized metal affinity chromatography (IMAC). Protein purity was determined by SDS-PAGE, and protein concentration was determined by measuring absorbance at 280 nm and combining it with the extinction coefficient (149090 M). -1 cm -1 )calculate.

[0038] β-N-acetylglucosaminease activity was assayed using p-nitrophenyl N-acetyl-β-D-glucosinolate (pNP-GlcNAc) as the substrate. The standard reaction system (500 μL) consisted of 100 μL of 10 mM 4-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside (pNP-GlcNAc, CAS No.: 3459-18-5), 350 μL of 50 mM sodium phosphate buffer (pH 6.5), and 50 μL of diluted enzyme solution. The reaction was carried out at 60°C for 5 minutes and terminated by adding 500 μL of 1 M sodium carbonate. The product, p-nitrophenol (pNP), was quantified by absorbance measurement at 405 nm. Enzyme activity units (U) are the amount of enzyme required to release 1 μmol of pNP per minute under the assay conditions. All assays were performed in triplicate. cbHEX exhibited optimal enzyme activity at 50°C and pH 6.5, but its activity decreased sharply at temperatures above 60°C or pH values ​​outside the 5.0–8.5 range. Figure 3 ).

[0039] HPLC analysis of the hydrolysis products showed that cbHEX could rapidly hydrolyze chitosan oligosaccharides with higher degrees of polymerization into oligosaccharides with lower degrees of polymerization within 5 min, proving its exonuclease activity. After 30 min, the chitosan oligosaccharides were completely hydrolyzed into N-acetylglucosamine (GlcNAc) as the final product. Figure 4 Differential scanning fluorescence (DSF) experiments showed that the melting temperature T of cbHEX was... m The temperature is 43.5℃, and the half-life (t) is... 1 / 2 At 40℃, it took 335 minutes; at 50℃, it took 4 minutes; and at 60℃, it took only 1 minute. Figure 5 This indicates that cbHEX has weak heat resistance and is difficult to maintain its activity under high temperature conditions for a long time.

[0040] The specific activity of this enzyme for pNP-GlcNAc was 872±47 U, for pNP-GalNAc it was 112±6 U, while no catalytic activity was detected for pNP-Glu and pNP-Gal. Figure 6 These results indicate that cbHEX is an exonuclease specific to GlcNAc / GalNAc, exhibiting high catalytic activity but limited thermal stability.

[0041] 10% (w / v, g / mL) suspension of crystalline chitin was prepared (50 mM sodium acetate buffer, pH 6.0), and 200 μg of 76Chi (also known as ChBD-CD) was added. chi (See SUN B, ZHAO XC, XU BR, et al. Discovering and Designing a Chimeric Hyperthermophilic Chitinase for Crystalline Chitin Degradation[J]. ACS Sustainable Chemistry & Engineering, 2023, 11(12): 4690-4698.) When acted alone on crystalline chitin for 3 h, 7.70 g L of the product was generated. -1 (GlcNAc)2; with the addition of 5 μg cbHEX, only 0.37 g L was generated. -1 GlcNAc, and still 7.53 g L -1 (GlcNAc)2 was not further hydrolyzed. Figure 9 Due to the poor thermal stability of cbHEX, the co-hydrolysis of chitin is severely limited. Therefore, further enzyme engineering modification of cbHEX is needed to improve its thermal stability and achieve efficient co-degradation of crystalline chitin.

[0042] Example 3: High-resolution crystal structure of cbHEX and structural evaluation using an AI model.

[0043] To obtain a high-quality structural template suitable for rational modification, this invention first resolved the X-ray crystal structure of cbHEX, and then used AlphaFold2 (AF2) and AlphaFold3 (AF3) to predict its structure, so as to compare the reliability of the experimental structure and the AI-predicted structure in subsequent protein design.

[0044] According to the NCBI Conserved Domain Annotation, cbHEX consists of four domains: a carbohydrate-binding domain CBD (19-163), an α / β domain (183-308), a catalytic domain CD (311-728), and a C-terminal domain (765-832), connected by two flexible linker regions, Linker 1 (161-186) and Linker 2 (728-769). Figure 7 Among them, Loop A (454-481), Loop B (510-524), Loop D (567-587), Loop E (593-604), and Loop F (617-647) together constitute the core area of ​​the active pocket.

[0045] To evaluate the structural reliability of the crystal structure, the AF2 model, and the AF3 model, the MolProbability evaluation index of the three was systematically compared (Table 1). Ramachandran plot outlier ratio and dominant conformation analysis showed that all three structures had good main chain geometry quality, with over 98% of residues located in the favored region, and none exhibited outliers (Table 1). However, compared to the crystal structure, the AF predicted structure showed a higher clash score and a higher MolProbability score, indicating that there are still some errors in its overall atomic arrangement and local geometry. Specifically, the AF2 model had a bad bond angle ratio of 0.26% and Cα geometry outliers of 0.61%, both exceeding the recommended thresholds. Although the AF3 model outperformed AF2 in these indicators, its overall quality still did not reach the level of the crystal structure. In summary, although all three structures have good quality, the crystal structure remains the most reliable structural template, followed by AF3, while AF2 is relatively poor.

[0046] Table 1. Quality analysis of the X-ray crystal structure of cbHEX and the predicted structures of AF2 and AF3.

[0047]

[0048] Example 4: SDACS strategy for screening candidate mutation sites

[0049] To systematically screen candidate mutation sites with the potential to improve the thermal stability and catalytic activity of cbHEX, this embodiment employs the SDACS strategy for rational design, comprising three parts: Strategy 1, Strategy 2, and disulfide bond engineering. Specifically, the following steps are included:

[0050] Strategy 1 combines two dimensions: folding free energy change and evolutionary conservation. Using the X-ray crystal structure of cbHEX, the predicted structure of AF2, and the predicted structure of AF3 as templates, the folding free energy change (ΔG) is calculated using the Cartesian_ddg protocol in Rosetta software. fold Before energy calculation, the wild-type structure undergoes a two-step relaxation process: first, a coordinate-constrained FastRelax is applied, followed by Cartesian FastRelax optimization using the ref2015_cart energy function to obtain the energy-minimized conformation. ΔG for each mutation... fold according to Formula calculation, where This represents the folding free energy of the mutant. This represents the folding free energy of cbHEX wild-type. For each amino acid site in cbHEX, the remaining 19 natural amino acids were replaced, and virtual mutant libraries were constructed based on three structural models. Each library contained a total of 827 × 19 = 15,713 virtual single-point mutations. Then, sequence conservation analysis was performed on cbHEX. A total of 1800 homologous sequences (30–90% identity) were obtained and clustered into 200 representative sequences. Subsequently, the frequency of wild-type residues at each site was calculated using the HotSpot Wizard online server. WT ) and frequency of mutant residues (Fre mut Only Fre is retained. mut ≥ Fre WT mutations, while excluding Fre WT Greater than 10% of conserved residues are required to avoid disrupting important functional sites. Under more stringent screening conditions, candidate mutations must simultaneously satisfy ΔΔG. fold < -4.0 kcal mol -1 And Fre mut ≥ 6% (Table 2). Based on crystal structure, a total of 9 candidate mutations were screened under these conditions: T102Y, I144F, H210Y, A228L, S520A, T556V, G648K, P719A, and G764I; 11 candidate mutations were screened based on the AF2 model: T102Y, M124L, H210Y, S476V, A539T, P574K, P615V, H629Y, G648K, V718L, and P719A; and 7 candidate mutations were screened based on the AF3 model: G104E, M124L, H210Y, H629Y, G648K, V718L, and T737D. The ΔΔG value was slightly relaxed. fold The filtering condition is -4.0 ≤ ΔΔG. fold<-3.0 kcal mol -1 Furthermore, the conservation constraint was strengthened to require that candidate mutations have the highest frequency at the site. Additional candidate mutations were obtained: four new candidate mutations were added based on the crystal structure, namely T120V, S476V, H629Y, and A711Y; four new candidate mutations were added based on the AF2 model, namely G104E, M511F, T516V, and T556L; and five new candidate mutations were added based on the AF3 model, namely A228L, M511F, T516V, A539T, and P615V. It is noteworthy that six mutations were repeatedly identified in different structural templates, including H629Y (X-ray / AF2 / AF3), G648K (X-ray / AF2 / AF3), T102Y (X-ray / AF2), A228L (X-ray / AF3), M511F (AF2 / AF3), and T516V (AF2 / AF3), indicating that different structural models are consistent in predicting candidate sites, and also show a certain degree of complementarity.

[0051] Table 2 Strategy 1: Based on X-ray crystal structure, AF2 model and AF3 model, design mutations using sequence conservation and folding free energy difference.

[0052]

[0053] Strategy 2 focuses on targeted design for highly flexible regions. The average B-factor of each residue in the cbHEX crystal structure is calculated using the BAVERAGE program in the CCP4 software package. The top 25% of residues are selected as candidate sites for high flexibility, and further screening is conducted to ensure they meet the ΔΔG criteria. fold ≤ -2.5 kcal·mol -1 And Fre mut Fre WT Mutations were identified (Table 3). Under this strategy, six candidate mutations were screened: A29L, T102Y, T120V, D169K, D805T, and S803R. These mutations are expected to improve the overall thermal stability of the protein by reducing local fluctuations in flexible regions and enhancing structural rigidity.

[0054] Table 3 Strategy 2: Based on crystal structure, designing mutations using sequence conservation, folding free energy difference, and B factor.

[0055]

[0056] Table 4. Disulfide Engineering: Disulfide by Design 2 - Predicting Possible Disulfide Bonds in cbHEX

[0057]

[0058] Disulfide bond engineering, as the third component of the SDACS strategy, is used to further improve the stability of flexible regions. Disulfide bond design is performed using the Disulfide by Design 2 (DbD2) online server. First, candidate residues are screened to ensure they meet geometric constraints: χ 3 The dihedral angle needs to be within ±5° of -87° or +97°. A comprehensive evaluation was conducted, combining parameters such as disulfide bond formation energy and local B-factor, to screen candidate sites suitable for introducing disulfide bond constraints into highly flexible regions (Table 4). Among all candidate residue pairs, S557C / E584C exhibited the optimal design parameters, with the lowest predicted formation energy of 0.25 kcal / mol. -1 The sum of the B factors of the two residues is 75.23 Å. 2 This indicates that the region has high flexibility; meanwhile, its predicted χ² value... 3 The angle is +95.66°, which is within a reasonable range suitable for disulfide bond formation. Based on the above results, this study selected S557C / E584C as the design site for disulfide bond engineering for subsequent experimental verification.

[0059] Example 5: Evaluation of the activity and thermal stability of the designed mutant

[0060] Table 5. Activity and thermal stability analysis of beneficial mutants

[0061]

[0062] Based on the calculation and screening results of Example 4, each candidate mutant was constructed and expressed, and its catalytic activity and thermal stability were experimentally verified. First, the relative activity and melting temperature (T0) of each mutant at 60°C were determined. m This will simultaneously satisfy the conditions of increased relative activity and T. m The improved mutants were defined as beneficial mutants, and their half-lives at 50°C were further determined (Table 5). The experimental results showed that 13 single-point mutants simultaneously improved activity and thermal stability, namely T102Y, T120V, I144F, S520A, G764I, H629Y, G648K, A228L, M511F, T516V, A29L, D169K, and S803R. In addition, the disulfide bond mutant S557C / E584C also showed good improvement in thermal stability.

[0063] Among the aforementioned single-point beneficial mutations, G764I showed the most significant overall improvement, with its relative activity increasing by 343%. mIncreasing the temperature by 8.2℃ prolonged the half-life to 11.3 min at 50℃. T516V also showed significant advantages over M511F: the relative enzyme activity of T516V increased to 199%, and T... m Increasing the temperature by 8.5℃ prolonged the half-life to 7.3 min; the relative enzyme activity of M511F increased to 134%, T m Increasing the temperature by 8.3°C extends the half-life to 8.5 min. The T of S803R... m The temperature was only slightly increased by 0.2℃, but its half-life was extended to 24.2 min, the longest among all single-point mutants; the half-life of D169K was also significantly extended to 16.2 min, and the relative enzyme activity increased to 114%. m Increased by 0.9℃; the relative enzyme activity of T102Y increased to 126%, T m Increasing the temperature by 0.9℃ extended the half-life to 7.4 min; the relative enzyme activity of A29L increased to 445%, and T... m The half-life was increased by 4.5 °C to 5.1 min. These results indicate that structural models from different sources can identify beneficial mutation sites, but the success rates vary. Further analysis shows that these beneficial mutations are not all predicted by the same structural model. Specifically, S803R, D169K, G764I, and A29L are beneficial mutations predicted solely by crystal structure; T102Y was predicted by both crystal structure and the AF2 model; and M511F and T516V were predicted by both the AF2 and AF3 models. These results demonstrate that crystal structure and AlphaFold predicted structures are significantly complementary in identifying beneficial mutations.

[0064] In addition to the single-point mutations mentioned above, the T-type mutation in the disulfide bond mutant S557C / E584C... m The largest increase was observed at 8.9℃, with a relative enzyme activity increasing by 121% and a half-life extended to 14.0 min, indicating that the introduction of disulfide bonds effectively enhances the enzyme's thermostability. While H629Y, I144F, G648K, A228L, S520A, and T120V also showed some improvement in activity and thermostability, their half-lives at 50℃ were only 3.6–4.3 min, offering limited improvement over the wild type, and therefore were not included in subsequent combined mutant designs.

[0065] Based on the above experimental results, A29L, T102Y, D169K, G764I, S803R, M511F, T516V and S557C / E584C were finally selected as candidate mutations for subsequent combination design to further improve the thermostability and catalytic activity of β-N-acetylglucosaminease. Among them, the mutant containing the A29L single-point mutation is named A29L, and its amino acid sequence is SEQ ID NO:3; the mutant containing the T102Y single-point mutation is named T102Y, and its amino acid sequence is SEQ ID NO:4; the mutant containing the D169K single-point mutation is named D169K, and its amino acid sequence is SEQ ID NO:5; the mutant containing the G764I single-point mutation is named G764I, and its amino acid sequence is SEQ ID NO:6; the mutant containing the S803R single-point mutation is named S803R, and its amino acid sequence is SEQ ID NO:7; the mutant containing the M511F single-point mutation is named M511F, and its amino acid sequence is SEQ ID NO:8; the mutant containing the T516V single-point mutation is named T516V, and its amino acid sequence is SEQ ID NO:9; the mutant containing the S557C and E584C disulfide bond mutations is named S557C / E584C, and its amino acid sequence is SEQ ID NO:9. NO:10.

[0066] Example 6: Stepwise Combination Strategy of Beneficial Mutations

[0067] Among the beneficial mutations verified experimentally, S803R, D169K, G764I, T102Y, and A29L were beneficial mutations obtained based on crystal structure screening, while M511F and T516V were beneficial mutations obtained based on the AF2 / AF3 model. To evaluate the synergistic effect among crystal structure-derived mutations, ΔΔG was first performed on all possible combinations of the five sites A29L, T102Y, D169K, G764I, and S803R. fold Calculations were performed. The results showed that as the number of combined mutations increased, ΔΔG... fold The values ​​gradually decrease, with the combination M5, which contains all five mutations, having the lowest predicted ΔΔG. fold The value is -22.6 kcal·mol⁻¹ -1 This indicates that it has good potential in improving thermal stability (Table 6).

[0068] Subsequently, the predicted multi-point combination mutants were constructed, expressed, and purified. Mutants M2 (containing two-point mutations of T102Y and D169K, amino acid sequence SEQ ID NO:11), M3 (containing multiple-point mutations of T102Y / D169K / G764I, amino acid sequence SEQ ID NO:12), M4 (containing A29L / T102Y / D169K / G764I, amino acid sequence SEQ ID NO:13), and M5 (containing multiple-point mutations of A29L / T102Y / D169K / G764I / S803R, amino acid sequence SEQ ID NO:14) were obtained. Experimental results showed that M5 exhibited the best overall performance among the above combinations, with its T... m The optimal reaction temperature was increased to 53.6℃, ​​10.1℃ higher than the wild type; the relative enzyme activity at 60℃ was increased to 6.2 times that of the wild type; the optimal reaction temperature increased from 50℃ to 55℃; and the half-life at 50℃ was extended to 30 min (Table 7). Further kinetic analysis showed that the K of M5... cat It decreased by about 3% compared to the wild type, but its K m The decrease from 0.37 mM to 0.30 mM indicates an improvement in substrate affinity; therefore, its catalytic efficiency (Km) is also improved. cat / K m Increased to 6532 ± 769 s -1 ·mM -1 (Table 7). The above results show that, through a reasonable combination of five beneficial mutations derived from the crystal structure, catalytic activity can be enhanced while improving thermal stability.

[0069] Table 6 Calculation of ΔΔG fold Guided combination of multiple mutations

[0070]

[0071] By further introducing the disulfide bond mutation S557C / E584C into M5, M7 was constructed, with its amino acid sequence SEQ ID NO:15. Experimental results show that the thermal stability of M7 is further enhanced, and its T m The temperature was increased to 58.4℃, 14.9℃ higher than the wild type; the half-life at 50℃ was extended to 92 min; and the optimal reaction temperature was increased to 60℃. Kinetic analysis showed that the Kt of M7 was... cat It is about 18% higher than M5, but K m It also increased by 20%, leading to a decrease in its catalytic efficiency (K). cat / K m The time decreased slightly to 6406 ± 611s. -1 mM -1(Table 7). The above results show that the introduction of disulfide bond engineering can significantly enhance the thermal stability of the mutant, but does not improve the catalytic efficiency.

[0072] Given that M511F and T516V are beneficial mutations specifically recognized near the active pocket in the AF2 / AF3 model, this invention further introduces these two mutations into M7 to construct the nine-mutant M9, whose amino acid sequence is SEQ ID NO:16. Experimental results show that the overall performance of M9 is further improved, and its T... m The optimal reaction temperature increased to 59.5℃, an increase of 16.0℃ compared to the wild type; the optimal reaction temperature increased from 50℃ in the wild type to 65℃. Figure 8 The half-life of M9 at 50°C increased from 4 min in the wild type to 240 min (Table 7). Kinetic analysis showed that the Kc of M9... m Significantly reduced to 0.16 mM, while K cat Increased to 4668.1 s -1 Therefore, its catalytic efficiency (K) cat / K m Increased to 29,314 ± 3,129 s -1 mM -1 The activity level was 5.4 times that of the wild type. These results indicate that the local conformational information of the active pocket provided by the AF2 / AF3 model can serve as an important supplement to the global stabilization design of crystal structures. Through synergistic combination with crystal structure-origin mutations and disulfide bond engineering, β-N-acetylhexosaminease mutants with both high thermal stability and high catalytic activity can be obtained.

[0073] At 65 °C, 5 μg M9 and 200 μg 76Chi were used for the synergistic hydrolysis of crystalline chitin. After 3 h of reaction, the chitobiose in the system was completely converted, and the yield of N-acetylglucosamine (GlcNAc) reached 9.88 g / L. -1 Compared to the cbHEX synergistic system, the M9 synergistic system increased monosaccharide yield by 26.7 times and chitin degradation rate by 28%. Figure 9 This indicates that the mutant constructed in this invention exhibits superior catalytic performance and application potential during the high-temperature synergistic enzymatic hydrolysis and crystallization of chitin.

[0074] Table 7 Comparison of the activity and thermal stability of cbHEX and its combined mutants

[0075]

[0076] 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 β-N-acetylhexosaminease mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

16.

2. The gene encoding the β-N-acetyxosaminease mutant of claim 1.

3. A recombinant vector comprising the gene of claim 2.

4. Recombinant engineered bacteria comprising the gene of claim 2 or the recombinant vector of claim 3.

5. The recombinant engineered bacteria according to claim 4, characterized in that, The recombinant engineered bacteria is Escherichia coli.

6. The use of the β-N-acetylglucosamine enzyme mutant of claim 1 in the synergistic exochitinase hydrolysis of chitin to prepare N-acetylglucosamine.

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