High-activity glucosamine synthetase and preparation and application thereof

Abstract

The application discloses a high-activity glucosamine synthetase and preparation and application thereof, and belongs to the technical field of enzyme engineering and biological manufacturing. In view of the problem that the activity of GlmS enzyme is impaired due to interference of a purification tag, a SUMO-GlmS fusion expression vector is constructed, a host cell is transformed to express a fusion protein, and after affinity purification, a tag is precisely removed by using a SUMO protease, so that the high-activity GlmS enzyme without the tag and in a natural conformation is obtained. The high-activity GlmS enzyme has an activity of 4.6 times higher than that of the same mutant with a His tag at a C terminal in catalyzing D-fructose and glutamine to generate glucosamine. The application also provides application of the enzyme to in-vitro high-efficiency synthesis of glucosamine. The application has simple process, high purification efficiency and significantly improved enzyme activity, and provides a new path for green, safe and high-efficiency biological manufacturing of glucosamine.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and protein design, biomanufacturing, and industrial biotechnology. Specifically, this invention relates to a method for preparing a highly active glucosamine synthase GlmS, the resulting highly active GlmS enzyme, and a method for efficiently synthesizing glucosamine in vitro using this enzyme as a substrate. Background Technology

[0002] Glucosamine is a key component of human cartilage matrix and synovial fluid. It has clear physiological functions in the prevention and treatment of osteoarthritis, cartilage repair and inflammation regulation, and has been widely used in medical preparations, functional foods and dietary supplements.

[0003] Currently, the industrial production of glucosamine mainly relies on two technological routes. The first is the traditional chemical extraction method, which involves preparing glucosamine from chitinous raw materials such as shrimp and crab shells through acid hydrolysis or enzymatic hydrolysis. This method is limited by marine resources, easily causes seafood allergic reactions, and also poses serious environmental pollution problems. The second is the microbial fermentation method, which uses metabolic engineering to construct cell factories to produce glucosamine. The core of this method lies in the regulation and optimization of glutamine-fructose-6-phosphate aminotransferase (GlmS). GlmS is a key rate-limiting enzyme in the glucosamine biosynthesis pathway, and its catalytic activity is strongly inhibited by the product glucosamine-6-phosphate and the final product glucosamine. At the same time, the intracellular glucosamine synthesis pathway competes with the glycolysis pathway for the common precursor fructose-6-phosphate, resulting in low carbon source allocation efficiency.

[0004] To address the aforementioned issues, Guan et al. published a study titled "Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine" in ACS Chemical Biology in 2024. This study employed a combined active site saturation test and iterative saturation mutagenesis strategy to perform directed evolutionary modification of the GlmS enzyme from Bacillus subtilis, obtaining a quadruple mutant, BsGlmS-BK19, containing four mutation sites: S596D / V597G / S347H / G299Q. This mutant exhibits 1736 times the catalytic activity for D-fructose compared to the wild type, enabling direct synthesis of glucosamine from D-fructose without the need for phosphorylation. Using this mutant as a whole-cell catalyst, D-fructose was converted to glucosamine within 6 hours with a conversion rate of 65.32%, while the wild type achieved only 0.31% conversion under the same conditions.

[0005] However, during the invention process, the inventors discovered significant shortcomings in the practical application of the aforementioned existing technologies. While this research yielded a highly active GlmS mutant, it failed to consider the potential interference of protein purification tags on enzyme activity. Due to the unique catalytic mechanism of GlmS, introducing protein purification tags at the amino or carboxyl terminus significantly interferes with the enzyme's spatial conformation and catalytic activity. Existing technologies generally rely on histidine tags for recombinant expression and purification, resulting in severely impaired enzyme activity and fundamentally limiting the efficiency of glucosamine production via enzymatic catalysis. Furthermore, tagged fusion proteins are susceptible to protease degradation in whole-cell fermentation systems, leading to insufficient process stability. Using whole-cell catalysis may result in endotoxin residues in the product, making it difficult to meet food safety standards for raw material production.

[0006] Therefore, there is an urgent need in this field to develop a technical solution that can obtain a label-free, native conformation, and highly active GlmS enzyme to achieve efficient, stable, and safe preparation of glucosamine. Summary of the Invention

[0007] Therefore, the present invention aims to provide a method for preparing a highly active glucosamine synthase to solve the problem of protein purification tags interfering with the spatial conformation and catalytic activity of GlmS enzyme in the prior art. By using a SUMO tag fusion expression and precise excision strategy, a tag-free, native conformation, highly active GlmS enzyme is obtained, thereby achieving efficient in vitro enzymatic synthesis of glucosamine.

[0008] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have provided a method for preparing highly active glucosamine synthase to solve the above-mentioned technical problems, comprising the following steps: a) Construct a fusion expression vector comprising a nucleotide sequence encoding a SUMO tag and a nucleotide sequence encoding a GlmS protein that are operably linked in sequence; b) Transform the fusion expression vector constructed in step a) into host cells to express the SUMO-GlmS fusion protein; c) Purify the SUMO-GlmS fusion protein; d) The purified SUMO-GlmS fusion protein was digested with SUMO protease to release the GlmS protein; e) Remove the SUMO tag and SUMO protease from the enzyme digestion system and collect the purified tagless GlmS protein.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to discover and solve the technical problem of protein purification tags severely interfering with the spatial conformation and catalytic activity of the GlmS enzyme. While existing technologies have yielded highly active GlmS quadruple mutants capable of directly utilizing D-fructose, they generally rely on histidine tags for recombinant expression and purification, resulting in severely impaired enzyme activity and limiting the efficiency of enzymatic synthesis of glucosamine. This invention innovatively employs a SUMO tag fusion expression strategy, successfully obtaining a highly active GlmS enzyme with its native conformation and no amino acid residues through precise sclerosing of the SUMO protease. Experimental data show that the tagless GlmS enzyme prepared in this invention achieves a yield of 9.28 μmol / mL in the catalytic conversion of D-fructose to glucosamine, which is 4.6 times higher than the same mutant version with a C-terminal histidine tag (2.03 μmol / mL), fully demonstrating the significant effect of tag sclerosing on enzyme activity. Simultaneously, this invention establishes an in vitro enzymatic synthesis system, avoiding the endotoxin residue problem that may exist in whole-cell catalysis methods, providing a superior technical route for the production of food-grade glucosamine. Furthermore, the method of this invention is simple in steps, has high purification efficiency, and good process stability, and has good prospects for industrial application, laying an important foundation for the efficient biomanufacturing of glucosamine and its derivatives.

[0010] Based on the above technical solution, the present invention can be further improved as follows: Further: In step a), the GlmS protein is derived from Bacillus subtilis, and the amino acid sequence of the GlmS protein contains four mutation sites: S596D, V597G, S347H and G299Q.

[0011] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: This invention introduces four mutation sites, S596D, V597G, S347H, and G299Q, to enable the GlmS enzyme to directly catalyze the synthesis of glucosamine using inexpensive D-fructose as a substrate. This, combined with the tagless expression strategy of this invention, ultimately yields a glucosamine synthase that possesses both an ideal substrate profile and the highest catalytic activity.

[0012] Based on the above technical solution, the present invention can be further improved as follows: Further: In step a), the SUMO tag is attached to the N-terminus of the GlmS protein.

[0013] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: By attaching a SUMO tag to the N-terminus of the GlmS protein, a native N-terminal conformation without any additional amino acid residues can be obtained after enzymatic cleavage, minimizing interference with the active site of GlmS and thus ensuring high catalytic activity of the enzyme.

[0014] Based on the above technical solution, the present invention can be further improved as follows: Further: In step e), the SUMO tag and SUMO protease are removed by affinity chromatography again, the flow-through is collected, and the tagless GlmS protein is obtained.

[0015] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: By performing affinity chromatography again, the SUMO tag and SUMO protease can be removed simultaneously in one step, thus obtaining high-purity tag-free GlmS protein in a simple and efficient manner.

[0016] The present invention also provides a highly active glucosamine synthase, which is prepared by the method described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The highly active glucosamine synthase provided by this invention is in a tagless natural conformation, and its catalytic activity is more than 4.6 times higher than that of the GlmS protein with the same amino acid sequence and a C-terminal histidine tag.

[0018] Preferably, the catalytic activity of the enzyme is more than four times that of the GlmS protein with the same amino acid sequence and a C-terminal histidine tag.

[0019] The present invention also provides the application of the aforementioned highly active glucosamine synthase in the preparation of glucosamine or its derivatives.

[0020] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: Using the highly active glucosamine synthase described in this invention for in vitro enzymatic synthesis can fully utilize the ultra-high catalytic activity brought about by its unlabeled natural conformation, significantly improve the synthesis efficiency and product safety of glucosamine, and overcome the dual defects of existing technologies, such as label interference with enzyme activity and the risk of endotoxin in whole-cell catalysis.

[0021] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, it includes the following steps: S1. Provide the aforementioned highly active glucosamine synthase; S2. Using D-fructose and glutamine as substrates, an enzyme-catalyzed reaction is carried out in a reaction system containing the highly active glucosamine synthase to generate glucosamine.

[0022] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: This invention establishes an in vitro enzyme catalysis system using a label-free, highly active GlmS enzyme, achieving efficient synthesis of glucosamine using D-fructose as a substrate, and completely avoiding the impact of endotoxin residues on food safety in whole-cell catalysis.

[0023] Based on the above technical solution, the present invention can be further improved as follows: Further: the pH of the reaction system described in step S2 is 7.0 to 8.0, and the temperature is 30°C to 40°C.

[0024] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: The above reaction conditions provide the optimal catalytic environment for GlmS enzyme, ensuring that the enzyme maintains the highest catalytic activity and stability in the in vitro reaction system, thereby maximizing the synthesis efficiency of glucosamine.

[0025] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the reaction system of the method also contains KCl.

[0026] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: The addition of KCl to the reaction system of this invention can maintain the ionic strength required for enzyme activity and stabilize the spatial conformation of the GlmS enzyme, thereby further improving the synthesis efficiency of glucosamine. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 These are SDS-PAGE electrophoresis images of GlmS protein expression and purification in Example 1 of this invention. Image A shows the expression and purification results of the GlmSBsu-4Mut-CHis protein carrying a C-terminal histidine tag; lane M is the reference, lane UI is before induction, lane I is after induction, lane S is the supernatant, lane P is the precipitate, lane F is flow-through, and lane E is elution. Image B shows the expression and purification results of the NHis-SUMO-GlmSBsu-4Mut fusion protein carrying an N-terminal histidine tag and a SUMO tag; lane M is the reference, lane UI is before induction, lane I is after induction, lane S is the supernatant, lane P is the precipitate, lane F is flow-through, and lane E is elution.

[0029] Figure 2 This is an SDS-PAGE electrophoresis image of the tagless GlmSBsu-4Mut protein obtained by digesting the SUMO fusion protein with ULP1 protease in Example 1 of this invention. Lane M is the reference lane.

[0030] Figure 3 This is a standard curve plotted using glutamic acid as a standard in Example 1 of the present invention.

[0031] Figure 4 This is a comparison chart of the yields of D-fructose to glucosamine catalyzed by different GlmS variants in Example 1 of the present invention. Detailed Implementation

[0032] The following description is based on specific embodiments.

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0034] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0035] Example 1

[0036] This embodiment describes the preparation and activity detection of highly active glucosamine synthase.

[0037] 1. Materials and Methods 1.1 Strains and Plasmids Escherichia coli DH5α was used for plasmid construction and amplification, and Escherichia coli BL21(DE3) was used for protein expression.

[0038] 1.2 Main Reagents Restriction endonucleases NcoI, XhoI, and NheI; T4 DNA ligase; high-fidelity DNA polymerase; DNA molecular weight standards; protein molecular weight standards; plasmid extraction kits and gel extraction kits; biochemical reagents such as isopropyl-β-D-thiogalactoside, D-fructose, glutamine, KCl, Tris, and imidazole; Ni-NTA agarose affinity chromatography medium; glutamate content detection kit; and SUMO protease ULP1 (the nucleotide sequence of its catalytic domain is shown in SEQ ID NO:1) were prepared in our laboratory. In this example, Bacillus subtilis strain 168 is a publicly available type strain.

[0039] 1.3 Primer Design and Synthesis Primers were designed based on the glmS gene sequence of Bacillus subtilis subsp. subtilis 168 and the multiple cloning site sequence of the pET28a vector for target gene amplification and site-directed mutagenesis. The site-directed mutagenesis primer sequences used are as follows: G299Q F: As shown in SEQ ID NO:2; G299Q R: As shown in SEQ ID NO:3; S347H F: As shown in SEQ ID NO:4; S347H R: As shown in SEQ ID NO:5; S596D, V597GR: As shown in SEQ ID NO:6.

[0040] 1.4 Construction of expression vector 1.4.1 Construction of the GlmS quadruple mutant expression vector pET28a-GlmSBsu-4Mut-CHis carrying a C-terminal His tag Using genomic DNA from *Bacillus subtilis* subsp. *subtilis* 168 as a template, the full-length coding sequence of the *glmS* gene was amplified by PCR. The amplified product and the empty pET28a vector were double-digested with NcoI and XhoI, respectively. The target fragment was recovered, ligated using T4 DNA ligase, and transformed into *E. coli* DH5α competent cells. The cells were plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Single colonies were picked for colony PCR identification and sequencing verification to obtain the recombinant plasmid pET28a-GlmSBsu-WT.

[0041] Using pET28a-GlmSBsu-WT as a template, four site-directed mutations (S596D, V597G, S347H, and G299Q) were introduced in three rounds using circular plasmid PCR. After each round of mutation, the mutants were transformed, screened, and sequenced for verification, ultimately yielding the quadruple mutant expression vector pET28a-GlmSBsu-4Mut-CHis carrying a C-terminal 6×His tag.

[0042] 1.4.2 Construction of the GlmS quadruple mutant fusion expression vector pET28a-NHis-SUMO-GlmSBsu-4Mut carrying the SUMO tag A nucleotide sequence encoding the SUMO tag was synthesized (its nucleotide sequence is shown in SEQ ID NO:7). Overlap PCR primers were designed to fuse the SUMO sequence with the GlmS gene sequence. The GlmS gene (its nucleotide sequence is shown in SEQ ID NO:8, and the encoded amino acid sequence is shown in SEQ ID NO:9) was amplified using Bacillus subtilis genomic DNA as a template. Simultaneously, the SUMO fragment was synthesized, and the SUMO-glmS fusion fragment was obtained through overlap PCR. The fusion fragment and the pET28a empty vector were double-digested with NheI and XhoI, respectively. The target fragment was recovered, ligated, transformed, screened, and sequenced for verification to obtain the recombinant plasmid pET28a-NHis-SUMO-GlmSBsu-WT.

[0043] Using pET28a-NHis-SUMO-GlmSBsu-WT as a template, the same circular plasmid PCR method as in 1.4.1 was used to gradually introduce four site-directed mutations: S596D, V597G, S347H, and G299Q, ultimately obtaining the SUMO fusion quadruple mutant expression vector pET28a-NHis-SUMO-GlmSBsu-4Mut carrying an N-terminal 6×His tag.

[0044] 1.5 Expression and purification of the target protein The correctly sequenced recombinant plasmids pET28a-GlmSBsu-4Mut-CHis and pET28a-NHis-SUMO-GlmSBsu-4Mut were transformed into Escherichia coli expression strain BL21(DE3) by chemical transformation, respectively, and plated on LB agar plates containing kanamycin and incubated overnight at 37°C.

[0045] Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin. Incubate overnight at 37°C with shaking at 160 rpm. Transfer 1% of the inoculum to 500 mL of LB liquid medium containing kanamycin and expand the culture at 37°C with 160 rpm until the OD600 is approximately 0.4-0.6. Add IPTG inducer to a final concentration of 0.3 mM and continue induction culture at 22°C with 160 rpm for 16-20 hours.

[0046] Collect bacterial cells by centrifugation at 8000 rpm for 10 minutes at 4℃, and discard the supernatant. Resuspend the bacterial cells in 30 mL of lysis buffer. The lysis buffer formulation is: 50 mM Tris-HCl, 500 mM NaCl, 40 mM imidazole, pH 7.6.

[0047] The bacterial resuspension was placed in an ice bath, and the cells were disrupted using an ultrasonic cell disruptor. The disruption conditions were: 300 W power, 3 seconds operation time, 5 seconds interval, and a total disruption time of 25 minutes. After disruption, the cells were centrifuged at 4°C and 13,000 rpm for 20 minutes, and the supernatant was collected.

[0048] Add the supernatant to a Ni-NTA agarose affinity chromatography column pre-equilibrated with lysis buffer, controlling the flow rate to ensure thorough binding of the sample and the medium. After loading, wash with 10-20 column volumes of lysis buffer to remove contaminating proteins. Finally, elute the target protein with elution buffer containing: 50 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 7.6. Collect the elution peaks in separate tubes, and determine protein purity and molecular weight by SDS-PAGE electrophoresis.

[0049] 1.6 Enzymatic digestion and secondary purification of SUMO fusion protein The purified NHIs-SUMO-GlmSBsu-4Mut fusion protein was diluted to an appropriate concentration with dialysis buffer. The dialysis buffer formulation was: 50 mM Tris-HCl, 150 mM NaCl, pH 7.6. SUMO protease ULP1 was added at a mass ratio of 1:100, and the protein was digested overnight at 16°C.

[0050] Small samples before and after enzyme digestion were subjected to SDS-PAGE electrophoresis to determine the digestion efficiency. The digestion products were then loaded again onto a Ni-NTA agarose affinity chromatography column, and the flow-through was collected. At this point, the tagless GlmSBsu-4Mut protein, after the SUMO tag was removed, did not bind to the Ni-NTA medium and flowed out directly; while the His-tagged SUMO tag and the His-tagged fused ULP1 both bound to the medium. The collected flow-through was concentrated, aliquoted, and stored at -80°C for later use.

[0051] 1.7 Protein Concentration Determination The concentration of purified proteins was determined using the Bradford method, and a standard curve was plotted using bovine serum albumin as a standard. The concentration of the purified GlmSBsu-4Mut-CHis protein was 1.25 mg / mL, and the concentration of the NHis-SUMO-GlmSBsu-4Mut fusion protein was 1.48 mg / mL. The concentration of the unlabeled GlmSBsu-4Mut protein obtained after secondary purification via enzymatic digestion was 0.86 mg / mL after concentration. SDS-PAGE grayscale analysis showed that the purity of each purified protein sample was greater than 90%.

[0052] 1.8 GlmS enzyme activity assay 1.8.1 Construction of the Glutamate Standard Curve Follow the instructions of the glutamate content detection kit to prepare glutamate standard solutions of different concentrations, measure the absorbance, and plot the glutamate concentration-absorbance standard curve.

[0053] 1.8.2 Enzyme-catalyzed reactions The reaction system consisted of 1 mL of the following components: 100 mM phosphate buffer (pH 7.6), 20 mM D-fructose, 25 mM KCl, 20 mM glutamine, and 1 nmol of purified target protein. The reaction system was incubated at 37°C for 4 hours. Three parallel reactions were performed for each sample.

[0054] After the reaction was completed, the amount of glutamic acid produced in the reaction system was determined according to the instructions of the glutamic acid content detection kit. Since one molecule of glucosamine is produced simultaneously with one molecule of glutamic acid, the amount of glucosamine synthesized can be directly calculated from the amount of glutamic acid produced.

[0055] 2. Results 2.1 Expression and purification results of GlmS protein SDS-PAGE electrophoresis results are as follows: Figure 1 As shown.

[0056] Figure 1 Figure A shows the expression and purification results of GlmSBsu-4Mut-CHis protein carrying a C-terminal His tag. Lanes UI represent the protein before induction and I represent the protein after induction, showing a clear protein band at the expected molecular weight position after induction. Lane S represents the supernatant and P represents the precipitate, indicating that the target protein mainly exists in the supernatant in a soluble form. Lane F represents flow-through and E represents elution, showing that after purification by Ni-NTA affinity chromatography, a high-purity GlmSBsu-4Mut-CHis protein with a molecular weight of approximately 67 kDa was obtained, consistent with the theoretical value.

[0057] Figure 1Figure B shows the expression and purification results of the NHis-SUMO-GlmSBsu-4Mut fusion protein carrying N-terminal His and SUMO tags. Lanes UI represent the protein before induction and I represent the protein after induction, showing a clear protein band at the expected molecular weight position after induction. Lane S represents the supernatant and lane P represents the precipitate, indicating that SUMO fusion significantly improves the soluble expression of the target protein. Lane F represents flow-through and lane E represents elution, showing that after purification by Ni-NTA affinity chromatography, a high-purity NHis-SUMO-GlmSBsu-4Mut fusion protein with a molecular weight of approximately 80 kDa was obtained, consistent with the theoretical value.

[0058] 2.2 Results of enzymatic digestion and secondary purification of SUMO fusion protein SDS-PAGE electrophoresis results are as follows: Figure 2 As shown. In the lane before enzyme digestion, a single band of the NHIs-SUMO-GlmSBsu-4Mut fusion protein is visible; in the lane after enzyme digestion, the intensity of the fusion protein band is significantly reduced, and two new bands appear, namely the SUMO tag and the GlmSBsu-4Mut protein; in the flow-through lane after enzyme digestion, a single GlmSBsu-4Mut protein band with a molecular weight of approximately 67 kDa is visible; in the eluted lane after enzyme digestion, the SUMO tag band and a small amount of residual fusion protein band are visible.

[0059] Gray-scale scanning analysis showed that the integrated gray-scale value of the NHis-SUMO-GlmSBsu-4Mut fusion protein band before digestion was 83,898,286. After digestion, this band almost disappeared, and the integrated gray-scale value approached 0. The integrated gray-scale value of the GlmSBsu-4Mut protein band that appeared after digestion was 67,877,991. These results indicate that the ULP1 protease has a high digestion efficiency for the SUMO fusion protein. The flow-through obtained after secondary purification contained a single, unlabeled GlmSBsu-4Mut protein, and its purity met the requirements for subsequent activity assays.

[0060] 2.3 Glutamic acid standard curve The absorbance of glutamic acid standard solutions of different concentrations was measured according to the kit instructions, and a standard curve was plotted as follows: Figure 3 As shown. The standard curve equation is: y = 0.5698x + 0.0786, R² = 0.9951, showing good linearity, and can be used for the quantitative detection of glutamate content in subsequent samples.

[0061] 2.4 Comparison of GlmS enzyme activities Two equimolar amounts (1 nmol) of two GlmS variants—GlmSBsu-4Mut-CHis protein carrying a C-terminal His tag and a tagless GlmSBsu-4Mut protein obtained by SUMO excision—were added to the reaction system for enzymatic reaction. The amount of glutamate produced was measured, and the results are as follows: Figure 4 As shown in the figure. Three parallel reactions were set up for each sample, and the results are expressed as averages. Repeated experiments showed that the data had good reproducibility.

[0062] The GlmSBsu-4Mut-CHis protein carrying a C-terminal His tag catalyzed 2.03 μmol / mL of glutamate production from D-fructose, which translates to a glucosamine yield of 2.03 μmol / mL. The untagged GlmSBsu-4Mut protein catalyzed 9.28 μmol / mL of glutamate production from D-fructose, also translating to a glucosamine yield of 9.28 μmol / mL. The glucosamine yield of the untagged GlmS variant was 4.6 times higher than that of the same mutant version carrying a C-terminal His tag.

[0063] This result fully demonstrates that the C-terminal His tag severely interferes with the catalytic activity of GlmS enzymes, while the native conformation tagless GlmS enzyme obtained by removing the tag after SUMO fusion expression can fully exert its high catalytic activity and significantly improve the synthesis efficiency of glucosamine.

[0064] Example 2

[0065] This example describes the effect of reaction conditions on GlmS enzyme activity.

[0066] Using the unlabeled GlmSBsu-4Mut protein prepared in Example 1 as a catalyst, the effect of different reaction conditions on enzyme activity was investigated.

[0067] 2.1 Effect of pH on enzyme activity Based on the enzyme-catalyzed reaction system of Example 1, phosphate buffer solutions with pH values ​​of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 were used respectively, with other conditions remaining unchanged. After incubation at 37°C for 4 hours, the amount of glutamate produced was measured. The results showed that GlmSBsu-4Mut maintained high activity in the pH range of 7.0-8.0, with the optimal pH being around 7.5.

[0068] 2.2 Effect of temperature on enzyme activity Based on the enzyme-catalyzed reaction system of Example 1, the enzyme was incubated at 25℃, 30℃, 35℃, 37℃, 40℃, and 45℃ for 4 hours, and the amount of glutamate produced was measured. The results showed that GlmSBsu-4Mut maintained high activity in the range of 30℃-40℃, with the optimal temperature being around 37℃.

[0069] 2.3 Effect of KCl concentration on enzyme activity Based on the enzyme-catalyzed reaction system of Example 1, 0, 10, 25, 50, and 100 mM KCl were added respectively, with other conditions remaining unchanged. After incubation at 37°C for 4 hours, the amount of glutamate produced was measured. The results showed that the enzyme activity was highest when 25 mM KCl was added, and excessively high concentrations of KCl had a certain inhibitory effect on enzyme activity.

[0070] The above results indicate that the tagless GlmS enzyme prepared in this invention can efficiently catalyze the conversion of D-fructose to glucosamine under mild reaction conditions, and has good prospects for industrial application.

[0071] Example 3

[0072] This embodiment describes the application of highly active glucosamine synthase in the preparation of glucosamine.

[0073] The tagless GlmSBsu-4Mut protein prepared in Example 1 was used for scale-up experiments under optimized reaction conditions. The reaction system consisted of 100 mL of the following: 100 mM phosphate buffer (pH 7.6), 100 mM D-fructose, 25 mM KCl, 100 mM glutamine, and 100 nmol of tagless GlmSBsu-4Mut protein. The reaction was carried out at 37°C with shaking for 8 hours, and samples were taken periodically to determine the amount of glucosamine produced.

[0074] The results showed that after 8 hours of reaction, the glucosamine concentration reached 42.6 mM, with a conversion rate of 42.6%. No obvious byproducts were observed during the reaction, and the product purity was high. These results further confirm the potential application value of the highly active GlmS enzyme prepared in this invention in the large-scale preparation of glucosamine.

[0075] In summary, this invention, through an innovative strategy of precise excision following SUMO tag fusion expression, successfully obtained a tag-free, natively conformation-highly active GlmS enzyme. Its activity in catalyzing the conversion of D-fructose to glucosamine is 4.6 times higher than that of the same mutant version carrying a C-terminal His tag. The method of this invention is simple, highly efficient in purification, and exhibits good process stability, providing a new technical pathway for the efficient, safe, and green biomanufacturing of glucosamine, and has promising prospects for industrial application.

[0076] In the description of this invention, it should be understood that "-" and "~" represent a range between two values, and this range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0077] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0078] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0079] In the description of this invention, the terms “about” or “approximately” are used to express approximate values ​​or ranges, allowing for a certain degree of error to ensure the flexibility and practicality of the description, while remaining within an acceptable range of error, with the maximum error not exceeding 10% of the corresponding value or range.

[0080] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing highly active glucosamine synthase, characterized in that, Includes the following steps: a) Construct a fusion expression vector comprising a nucleotide sequence encoding a SUMO tag and a nucleotide sequence encoding a GlmS protein that are operably linked in sequence; b) Transform the fusion expression vector constructed in step a) into host cells to express the SUMO-GlmS fusion protein; c) Purify the SUMO-GlmS fusion protein; d) The purified SUMO-GlmS fusion protein was digested with SUMO protease to release the GlmS protein; e) Remove the SUMO tag and SUMO protease from the enzyme digestion system and collect the purified tagless GlmS protein.

2. The method according to claim 1, characterized in that, In step a), the GlmS protein is derived from Bacillus subtilis, and the amino acid sequence of the GlmS protein contains four mutation sites: S596D, V597G, S347H, and G299Q.

3. The method according to claim 1, characterized in that, In step a), the SUMO tag is attached to the N-terminus of the GlmS protein.

4. The method according to any one of claims 1-3, characterized in that, In step e), the SUMO tag and SUMO protease are removed by affinity chromatography again, and the flow-through is collected to obtain tag-free GlmS protein.

5. A highly active glucosamine synthase, characterized in that, Prepared by the method described in any one of claims 1 to 4.

6. The highly active glucosamine synthase according to claim 5, characterized in that, The enzyme's catalytic activity is more than four times that of the GlmS protein with the same amino acid sequence and a C-terminal histidine tag.

7. The use of the highly active glucosamine synthase according to claim 5 or 6 in the preparation of glucosamine or its derivatives.

8. The application according to claim 7, characterized in that, Includes the following steps: S1. Provide the highly active glucosamine synthase as described in claim 5 or 6; S2. Using D-fructose and glutamine as substrates, an enzyme-catalyzed reaction is carried out in a reaction system containing the highly active glucosamine synthase to generate glucosamine.

9. The application according to claim 8, characterized in that, The reaction system described in step S2 has a pH of 7.0 to 8.0 and a temperature of 30°C to 40°C.

10. The application according to claim 9, characterized in that, The reaction system of the method also contains KCl.

Images