Sucrose phosphorylase mutant BaSP-P134A and application thereof in production of glucose-1-phosphoric acid

Abstract

The invention relates to the technical field of bioengineering, in particular to a sucrose phosphorylase mutant BaSP-P134A and an application of the sucrose phosphorylase mutant BaSP-P134A in production of glucose-1-phosphoric acid. The sucrose phosphorylase mutant BaSP-P134A is obtained by carrying out site-specific mutagenesis on amino acid at the 134 position of an amino acid sequence as shown in SEQ ID NO.2; the site-specific mutagenesis is that proline at the 134 site of the amino acid sequence as shown in SEQ ID NO. 2 is mutated into alanine. The sucrose phosphorylase mutant BaSP-P134A can be applied to the field of industrial production, G-1-P can be efficiently prepared, the production intensity is high, the catalytic effect on a high-concentration substrate is good, the enzyme shows better stability, efficient conversion of sucrose can be achieved, and a favorable basis is further provided for industrial application of sucrose phosphorylase.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a sucrose phosphorylase mutant BaSP-P134A and its application in the production of glucose-1-phosphate. Background Technology

[0002] Glucose-1-phosphate (G-1-P) is a monosaccharide phosphate ester formed by phosphorylation of glucose at the 1-position. It is a key intermediate in glucose metabolism pathways and is highly water-soluble. G-1-P plays a crucial role in various biochemical reactions, particularly in glycogen metabolism, starch synthesis, and the preparation of glycoside derivatives. It is an important functional substrate in pharmaceuticals, food fermentation, and enzyme-catalyzed synthesis. For example, in cellular energy metabolism and glycogen replenishment, G-1-P, as a direct precursor to UDP-glucose synthesis, promotes efficient cellular glucose metabolism. Simultaneously, G-1-P serves as an important donor for the synthesis of polysaccharides and oligosaccharides, and can be used in biomanufacturing to construct functional oligosaccharide or polysaccharide materials.

[0003] Naturally occurring G-1-P is present in extremely low quantities, and large-scale acquisition typically relies on artificial preparation. Chemical methods were an early strategy for G-1-P preparation, but traditional chemical synthesis routes suffer from complex steps, low yields, numerous byproducts, and difficult purification. In contrast, enzymatic methods can achieve highly selective and efficient G-1-P preparation, while also being environmentally friendly and easier to separate and purify. Therefore, developing an efficient enzymatic preparation process is of great significance for the industrial production of G-1-P. Recent studies have shown that phosphorylation-related enzymes (such as sucrose phosphorylase) can be used to prepare G-1-P with high catalytic strength, good adaptability to high substrate concentrations, and good stability. Therefore, sucrose phosphorylase enzymatic catalysis is a promising next-generation G-1-P preparation method with significant industrial potential.

[0004] However, while the enzymatic preparation of G-1-P using sucrose phosphorylase offers advantages such as high selectivity and mild conditions, it still faces significant limitations in industrial applications. Firstly, the low catalytic efficiency of natural sucrose phosphorylases necessitates high enzyme concentrations to achieve substantial yields, thus increasing production costs. Secondly, as a reversible phosphatase process, the reaction is limited by the concentrations of sucrose and inorganic phosphate, hindering the sufficient accumulation of G-1-P at equilibrium. Furthermore, the byproduct fructose not only inhibits product production but also complicates subsequent separation and purification. In addition, most sucrose phosphorylases lack stability and are susceptible to high temperatures and substrate concentrations, making them easily inhibited or inactivated in high-concentration sucrose systems. G-1-P itself is also sensitive to pH and temperature, further compressing the process window. Moreover, immobilization and continuous production technologies are still immature, making the large-scale production of G-1-P using sucrose phosphorylase a considerable challenge. Therefore, protein engineering and molecular modification are needed to improve the enzyme's catalytic efficiency and stability to enhance its industrial application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a sucrose phosphorylase mutant, BaSP-P134A, and its application in the production of glucose-1-phosphate, thereby addressing the problems existing in the prior art. The sucrose phosphorylase mutant provided by this invention can efficiently prepare G-1-P with high production intensity, exhibits good catalytic effect on high-concentration substrates, demonstrates better enzyme stability, and can achieve efficient conversion of G-1-P, further providing a favorable foundation for the industrial application of sucrose phosphorylase mutants.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a sucrose phosphorylase mutant BaSP-P134A, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] This invention provides a gene encoding the above-mentioned sucrose phosphorylase mutant BaSP-P134A.

[0008] The present invention provides a recombinant vector containing the above-mentioned genes.

[0009] The present invention provides a recombinant bacterium containing the above-mentioned genes.

[0010] The present invention provides the application of the above-mentioned sucrose phosphorylase mutant BaSP-P134A, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant bacteria in the preparation of glucose-1-phosphate.

[0011] This invention provides the application of the above-mentioned sucrose phosphorylase mutant BaSP-P134A, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant bacteria in improving the production efficiency of glucose-1-phosphate.

[0012] The present invention provides a method for improving the production efficiency of glucose-1-phosphate, comprising the step of using the above-mentioned sucrose phosphorylase mutant BaSP-P134A to catalyze the synthesis of glucose-1-phosphate from sucrose.

[0013] Optionally, in the process of synthesizing glucose-1-phosphate, sucrose is used as the substrate and a buffer solution with a pH of 8.0 is used as the reaction medium to form a reaction system, and the reaction is carried out at 37°C to obtain glucose-1-phosphate.

[0014] This invention provides the application of the above-mentioned sucrose phosphorylase mutant BaSP-P134A, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant bacteria in the preparation of products containing glucose-1-phosphate.

[0015] The present invention provides a product for producing glucose-1-phosphate, the product comprising the above-mentioned sucrose phosphorylase mutant BaSP-P134A, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant bacteria.

[0016] The present invention discloses the following technical effects: This invention provides a sucrose phosphorylase mutant, BaSP-P134A. Compared to wild-type sucrose phosphorylase (BaSP), the optimal catalytic conditions for the sucrose phosphorylase mutant BaSP-P134A remain unchanged, while the mutant exhibits optimal catalytic conditions at 37°C. max Nothing changed, but k m The enzyme exhibits lower binding affinity and significantly improved catalytic efficiency. Applying this mutant to the industrial production of G-1-P enables efficient and high-intensity production of G-1-P, demonstrates good catalytic performance with high substrate concentrations, and exhibits better enzyme stability, thus achieving efficient G-1-P conversion. This provides a favorable foundation for the industrial application of sucrose phosphorylase mutants. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The results are from TLC (thin-layer chromatography) detection. Figure 2The proton NMR spectrum obtained by high performance liquid chromatography after the reaction of sucrose catalyzed by the sucrose phosphorylase mutant BaSP-P134A is shown. Figure 3 The carbon spectrum obtained by high performance liquid chromatography after the reaction of sucrose catalyzed by the sucrose phosphorylase mutant BaSP-P134A is detected. Figure 4 This is a phosphorylation reaction catalyzed by the sucrose phosphorylase mutant BaSP-P134A. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The experimental materials and reagents used in the embodiments of this invention are as follows: (1) The tryptone and yeast extract used in this invention were purchased from Oxoid Ltd., UK, and the remaining analytical and pure chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0025] (2) E. coli BL21 (DH5α), E. coli BL21 (DE3) and other molecular biology reagents were provided by Beijing TransGen Biotech Co., Ltd.

[0026] (3) Primer synthesis and sequencing services were provided by Nanjing Branch of Beijing Qingke Biotechnology Co., Ltd.

[0027] (4) The culture medium formula used is as follows: LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone and 10 g / L NaCl, with deionized water as solvent.

[0028] TB medium: 5 g / L glycerol, 13.33 g / L tryptone, 26.67 g / L yeast extract, 2.31 g / L KH2PO4 and 16.43 g / L K2HPO4·3H2O, with deionized water as solvent.

[0029] The sequences involved in the embodiments of the present invention are as follows: The optimized nucleotide sequence of the BaSP gene is shown in SEQ ID NO.1:

[0030] The optimized amino acid sequence encoded by the BaSP gene is shown in SEQ ID NO.2: MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEE DLAGIYRPRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTDSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDF ALPPLLLHALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYV GALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVALE.

[0031] The amino acid sequence of the sucrose phosphorylase mutant BaSP-P134A is shown in SEQ ID NO.3: MKNKVQLITYADRLGDGTIKSMTDILRTRFDGVYDGVHILPFFTPFDGADAGFDPIDHTKVDERLGSWDDVAELSKTHNIMVDAIVNHMSWESKQFQDVLAKGEESEYYPMFLTMSSVFPNGATEEDLAG IYRaRPGLPFTHYKFAGKTRLVWVSFTPQQVDIDTSDKGWEYLMSIFDQMAASHVSYIRLDAVGYGAKEAGTSCFMTPKTFKLISRLREEGVKRGLEILIEVHSYYKKQVEIASKVDRVYDFALPPLLL HALSTGHVEPVAHWTDIRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEDVDNLVNTIHANTHGESQAATGAAASNLDLYQVNSTYYSALGCNDQHYIAARAVQFFLPGVPQVYYVGALAGKNDMELLRKTNNGRDINRHYYSTAEIDENLKRPVVKALNALAKFRNELDAFDGTFSYTTDDDTSISFTWRGETSQATLTFEPKRGLGVDNTTPVAMLEWEDSAGDHRSDDLIANPPVVALE; where the lowercase part is the mutation part.

[0032] Example 1: Construction and expression of wild-type sucrose phosphorylase gene Sucrose phosphorylase (BaSP, GenBank IDAF543301.1) derived from *Bifidobacterium adolescentis* was synthesized by Suzhou Genewise Biotechnology Co., Ltd. after codon optimization. The sequence is shown in SEQ ID NO.1. The amino acid sequence encoded by the optimized gene sequence is shown in SEQ ID NO.2. The synthesized gene sequence was then transformed into the vector pET-28a(+) via the restriction enzyme sites BamHI and XhoI to synthesize the recombinant plasmid pET-28a(+)-BaSP. The synthesized recombinant plasmid was transformed into *E. coli* BL21(DE3) and plated on LB agar plates containing a final concentration of 100 μg / mL kanamycin. After incubation at 37°C for 12 h, recombinant *E. coli* strains stably expressing the BaSP gene were obtained. The recombinant Escherichia coli was named Escherichia coli Sucrose phosphorylase Bifidobacterium dentium UM001 and deposited on July 25, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:66764.

[0033] The enzyme production process of *E. coli* Sucrose phosphorylase Bifidobacterium dentium UM001 is as follows: Single colonies of *Escherichia coli* *Sucrose phosphorylase Bifidobacterium dentium* UM001 were picked and inoculated into 50 mL LB liquid medium containing 50 μg / mL kanamycin. The culture was incubated at 37°C and 220 rpm for 12 h. Then, the colonies were transferred to TB medium at a 1% (v / v) inoculation rate and incubated at 37°C and 220 rpm for 5 h. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 50 μg / mL, and the culture was induced at 25°C and 220 rpm for 18 h. The induced bacterial culture was centrifuged at 8000 rpm for 15 min, the supernatant was discarded, and 20 mL of 20 mM MES buffer (pH 8.0) was added to the precipitate (bacterial sediment). The mixture was shaken repeatedly until the bacterial sediment dissolved. Subsequently, the cells were ultrasonically disrupted at 400 W for 20 min (working for 2 s, then pausing for 3 s). The disrupted cells were then centrifuged at 8000 rpm for 15 min, and the supernatant was the wild-type sucrose phosphorylase BaSP enzyme solution.

[0034] Example 2 Construction and expression of mutant genes Using pET-28a(+)-BaSP as a template, primers were designed for site-directed mutagenesis. Based on the amino acid sequence encoded by the BaSP gene (SEQ ID NO.2), the mutant plasmid pET-28a(+)-P134A containing the sucrose phosphorylase mutant BaSP-P134A was constructed. The amino acid sequence of the sucrose phosphorylase mutant BaSP-P134A is shown in SEQ ID NO.3. Subsequently, the mutant plasmid pET-28a(+)-P134A was transformed into *E. coli*.

[0035] The designed primers are: P134A F: GCGGGCATTTATCGCGCTCGCCCGGGCCTG (SEQ ID NO. 4); P134A R: ATAATGGGTAAACGGCAGGGCCCGGGCGGCT (SEQ ID NO. 5).

[0036] PCR reaction system: 0.5 μL forward primer, 0.5 μL reverse primer, 0.5 μL template, 12.5 μL 2×TransStartFastPfu Fly PCR Super Mix, 11 μL ddH2O, total volume 25 μL.

[0037] PCR amplification conditions: 94℃ pre-denaturation for 5 min, (94℃ for 20 s, 55℃ for 30 s, 72℃ for 90 s) for 25 cycles, 72℃ for 10 min, and finally incubation at 4℃.

[0038] Point mutation was first performed using the original enzyme plasmid pET-28a(+)-BaSP as a template. Using the primer pair (P134A F and P134A R), PCR amplification was carried out under the aforementioned conditions to construct the mutant plasmid. Agarose gel electrophoresis was used for identification. Correctly identified PCR products were excised and recovered to obtain the PCR amplification product. 0.5 μL of DMT enzyme was added to the PCR product and reacted at 37℃ for 1 h to digest the template, yielding the reaction mixture. The reaction mixture was then transformed into E. coli BL21 (DH5α) and plated on LB agar plates containing a final concentration of 50 μg / mL kanamycin, and incubated at 37℃ for 12 h. Single clones were picked from the plates by Beijing Qingke Biotechnology Co., Ltd. Nanjing Branch for culture, plasmid extraction, and sequencing. After sequencing verification, the mutant plasmid pET-28a(+)-P134A was finally obtained. The successfully sequenced mutant plasmid pET-28a(+)-P134A was transformed into E. coli BL21(DE3) and plated on LB agar plates containing 100 μg / mL kanamycin. The plates were then incubated at 37°C for 12 h. The enzyme production process was then performed according to the method described in Example 1 to obtain the sucrose phosphorylase mutant BaSP-P134A enzyme solution.

[0039] Example 3: Enzyme activity and specific activity assays of wild-type sucrose phosphorylase BaSP and the sucrose phosphorylase mutant BaSP-P134A. Enzyme activity assay conditions: Total volume 1 mL, enzyme addition 200 μL, 50 mM disodium hydrogen phosphate and 50 mM sucrose added, reacted at 37 ℃ and pH 8.0 for 1 h. After the reaction, 50 μL of the reaction solution was added to 200 μL of water and 750 μL of DNS and boiled at high temperature for 5 min. Enzyme activity was determined by DNS method.

[0040] Enzyme activity is defined as the amount of enzyme required to produce 1 μmol of fructose per minute, which is 1 U.

[0041] The formula for calculating enzyme activity is as follows: .

[0042] Table 1. Crude enzyme activities of wild-type sucrose phosphorylase BaSP and sucrose phosphorylase mutant BaSP-P134A Example 4 Kinetic constants of wild-type sucrose phosphorylase BaSP and sucrose phosphorylase mutant BaSP-P134A Different sucrose concentration gradients were set up (100 mM, 200 mM, 300 mM, 400 mM, 500 mM and 600 mM), 1 mL of pure enzyme solution was added, and the reaction was carried out at 37℃ and pH 8.0 for 5-10 min. The enzyme activity was determined by the DNS method, and the protein concentration was determined by the Bradford method. The specific activity was then calculated.

[0043] Using Origin to fit the Michaelis equation, K was further calculated. m V max k cat and k cat / K m The experimental results are shown in Table 2, V max and k cat The unchanged value indicates that the enzyme's catalytic ability remains unchanged. The K+ of the sucrose phosphorylase mutant BaSP-P134A... m The value decreased compared to the original enzyme, k cat / K m The increase indicates an enhanced affinity for the substrate and a significant improvement in catalytic efficiency.

[0044] Table 2 Kinetic constants of wild-type sucrose phosphorylase BaSP and sucrose phosphorylase mutant BaSP-P134A Example 5: Synthesis of G-1-P catalyzed by the sucrose phosphorylase mutant BaSP-P134A The phosphorylation reaction catalyzed by the sucrose phosphorylase mutant BaSP-P134A leads to the synthesis of G-1-P via the following pathway: Figure 4 As shown, using sucrose (Suc, composed of blue glucose α2 and red fructose) and inorganic phosphate (P) as substrates and the sucrose phosphorylase mutant BaSP-P134A as catalyst, Glc-1-P (blue glucose linked to phosphate groups) and fructose (Fru, red) were finally obtained.

[0045] Reaction conditions: Total volume 1 mL, enzyme addition 200 μL, 50 mM disodium hydrogen phosphate and 50 mM sucrose added, reacted at 37℃ and pH 8.0 for 1 h to obtain the enzyme-catalyzed reaction solution. After the reaction, the products in the reaction solution were detected by TLC (developing solvent: n-butanol:acetic acid:water = 4:1:1). Sucrose solution and glucose-1-phosphate solution with a concentration of 0.02 g / mL were used as standards. The experimental results are shown in [Figure number missing]. Figure 1 HPLC external standard method analysis showed that the yield of G-1-P synthesized by the wild-type catalysis was only 32.7%, while the yield of G-1-P synthesized by the mutant catalysis was significantly increased to 70.8%. The enzymatic product was purified by dextran gel G-15 packing material, and the product structure identification results are as follows. Figure 2 and Figure 3 As shown.

[0046] The results showed that the structure of the product was: 1 H NMR (600 MHz, D2O) δ 5.29 (dd, J = 7.4, 3.5Hz, 1H), 3.77 (ddd, J = 10.1, 5.3, 2.3 Hz, 1H), 3.71 (dd, J = 12.3, 2.3 Hz,1H), 3.65 – 3.55 (m, 2H), 3.31 (ddd, J = 9.8, 3.5, 1.8 Hz, 1H), 3.23 (dd, J =10.2, 9.2 Hz, 1H). 13 C NMR (151 MHz, DO) δ 93.54, 93.50, 73.14, 72.23, 72.19, 71.93, 69.74, 60.69.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A sucrose phosphorylase mutant BaSP-P134A, characterized in that, The amino acid sequence of the sucrose phosphorylase mutant BaSP-P134A is shown in SEQ ID NO.

3.

2. A gene encoding the sucrose phosphorylase mutant BaSP-P134A of claim 1.

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

4. A recombinant bacterium comprising the gene of claim 2.

5. The use of the sucrose phosphorylase mutant BaSP-P134A according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant bacteria according to claim 4 in the preparation of glucose-1-phosphate.

6. The application of the sucrose phosphorylase mutant BaSP-P134A according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant bacteria according to claim 4 in improving the production efficiency of glucose-1-phosphate.

7. A method for improving the production efficiency of glucose-1-phosphate, characterized in that, The method includes the step of using the sucrose phosphorylase mutant BaSP-P134A as described in claim 1 to catalyze the synthesis of glucose-1-phosphate from sucrose.

8. The method according to claim 7, characterized in that, In the process of synthesizing glucose-1-phosphate, sucrose is used as the substrate and a buffer solution with a pH of 8.0 is used as the reaction medium to form a reaction system. The reaction is carried out at 37°C to obtain glucose-1-phosphate.

9. The use of the sucrose phosphorylase mutant BaSP-P134A according to claim 1, the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant bacteria according to claim 4 in the preparation of products producing glucose-1-phosphate.

10. A product for producing glucose-1-phosphate, characterized in that, The product includes the sucrose phosphorylase mutant BaSP-P134A of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant bacteria of claim 4.

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