Difunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutants and uses thereof

Abstract

The application discloses a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. The application provides a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant, wherein at least one of the glutamic acid at the 28th position, the arginine at the 83rd position, the valine at the 126th position, the isoleucine at the 161st position, the leucine at the 187th position, the phenylalanine at the 249th position, the leucine at the 319th position and the tyrosine at the 343rd position in the amino acid sequence shown in SEQ ID No. 1 is mutated. The application further improves the conversion efficiency of D-mannose, and overcomes the problem of a large amount of by-products.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering and biocatalysis technology, specifically relating to bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutants and their applications. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] D-Mannose, as a functional sugar, possesses various physiological activities such as regulating the immune system, anti-inflammation, anti-tumor, and antibacterial properties, and has been widely used in medicine, food, and daily chemical industries. With the rapid development of emerging industries such as functional foods, precision medicine, and green feed, the market demand for D-Mannose continues to rise, indicating broad development prospects and enormous application potential.

[0004] Currently, the preparation of D-mannose mainly relies on two routes: one is direct extraction from plants or microorganisms, but this suffers from low extraction rates and high energy consumption; the other is chemical isomerization using glucose as a substrate, but this method produces many byproducts and involves complex purification processes. In recent years, enzymatic catalysis has become a research hotspot due to its green and efficient characteristics. However, traditional isomerases (such as glucose isomerase) are limited by thermodynamic equilibrium, and the D-mannose conversion rate is usually below 25%. In contrast, using maltodextrin as a substrate, the synergistic action of α-glucan phosphorylase (αGP), glucose phosphate mutase (PGM), the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant (PGI / PMI), and mannose-6-phosphate phosphatase (M6PP) can achieve highly efficient synthesis of D-mannose. To improve conversion efficiency, existing research has optimized some key enzymes. For example, a patented technology provides a method to enhance the conversion of mannose-6-phosphate to D-mannose by increasing the activity of M6PP through site mutation. However, the preceding step in this pathway, the conversion of glucose-6-phosphate to mannose-6-phosphate, is primarily catalyzed by PGI / PMI, and its catalytic efficiency directly affects the overall reaction process. Currently, research on protein engineering modifications of PGI / PMI is still relatively scarce, and there are no reports of obtaining highly active mutants through rational design or directed evolution. Therefore, it is urgent to optimize the catalytic performance of PGI / PMI through enzyme molecular modification techniques to significantly improve the bioconversion efficiency of maltodextrin to D-mannose.

[0005] In addition, existing multi-enzyme cascade catalytic systems based on maltodextrin have significant technical bottlenecks: on the one hand, the reaction pathway is long and the enzyme system is complex; on the other hand, a large amount of byproducts such as glucose and fructose are generated, resulting in low yield of the target product and difficulty in separation and purification, which seriously restricts the economic efficiency and feasibility of industrial production of D-mannose.

[0006] The existence of these key issues makes the development of efficient and highly specific bifunctional enzyme catalysts a current research focus. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a bifunctional enzyme glucose phosphoisomerase / mannose phosphoisomerase mutant and its applications. This invention aims to improve the conversion efficiency of D-mannose while overcoming the problem of a large number of byproducts.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] As a first aspect of the invention, a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant is provided, wherein the amino acid sequence of the mutant is mutated at at least one of the following positions: glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0010] Compared to the wild-type enzyme with an amino acid sequence as shown in SEQ ID NO.1, the single-point mutations of the mutant amino acids are selected from at least one of the following:

[0011] (1) The glutamic acid at position 28 is mutated to serine or aspartic acid;

[0012] (2) The arginine at position 83 is mutated to lysine or tryptophan;

[0013] (3) The valine at position 126 is mutated to isoleucine or phenylalanine;

[0014] (4) The isoleucine at position 161 is mutated to tryptophan or valine;

[0015] (5) The leucine at position 187 is mutated to methionine or isoleucine;

[0016] (6) The phenylalanine at position 249 is mutated to isoleucine or tyrosine;

[0017] (7) The leucine at position 319 is mutated to phenylalanine or tryptophan;

[0018] (8) The tyrosine at position 343 is mutated to phenylalanine or methionine.

[0019] Preferably, the amino acid of the mutant contains a combination of mutations at any 2 to 8 sites in the above-mentioned unit sites, which significantly improves the catalytic efficiency of the enzyme through a synergistic effect.

[0020] Preferably, it is selected from at least one of the following:

[0021] (1) Mutations exist simultaneously in glutamic acid at position 28 and arginine at position 83;

[0022] (2) Mutations exist simultaneously in glutamic acid at position 28 and valine at position 126;

[0023] (3) Mutations exist simultaneously in glutamic acid at position 28 and isoleucine at position 161;

[0024] (4) Mutations exist simultaneously in glutamic acid at position 28 and leucine at position 187;

[0025] (5) Mutations exist simultaneously in glutamic acid at position 28 and phenylalanine at position 249;

[0026] (6) Mutations exist simultaneously in glutamic acid at position 28, leucine at position 319, and tyrosine at position 343;

[0027] (7) Mutations exist simultaneously in glutamic acid at position 28, arginine at position 83, and tyrosine at position 343;

[0028] (8) Mutations exist simultaneously in arginine at position 83, isoleucine at position 161, and phenylalanine at position 249;

[0029] (9) Mutations exist simultaneously in the isoleucine mutation at position 161, the leucine mutation at position 187, and the phenylalanine mutation at position 249;

[0030] (10) Mutations exist simultaneously in glutamic acid at position 28, arginine at position 83, valine at position 126, and isoleucine at position 161;

[0031] (11) Mutations exist simultaneously in glutamic acid at position 28, arginine at position 83, valine at position 126, and tyrosine at position 343;

[0032] (12) Mutations exist simultaneously in arginine at position 83, isoleucine at position 161, phenylalanine at position 249, and tyrosine at position 343.

[0033] (13) Mutations exist simultaneously in arginine at position 83, valine at position 126, phenylalanine at position 249, and tyrosine at position 343;

[0034] (14) Mutations exist simultaneously in glutamic acid at position 28, valine at position 126, isoleucine at position 161, leucine at position 187 and phenylalanine at position 249.

[0035] (15) Mutations exist simultaneously in arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187 and phenylalanine at position 249.

[0036] (16) Mutations exist simultaneously in arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, leucine at position 319 and tyrosine at position 343.

[0037] (17) Mutations exist simultaneously in arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319 and tyrosine at position 343.

[0038] (18) Mutations exist simultaneously in the following positions: glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0039] As a second aspect of the invention, it is provided that a gene encoding a mutant of the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase described in the first aspect is provided.

[0040] As a third aspect of the invention, it is to provide biological material comprising the gene described in the second aspect, said biological material comprising an expression cassette, a vector, or a host cell; said host cell being selected from Escherichia coli and Bacillus subtilis.

[0041] As a fourth aspect of the invention, it is to provide the use of the biomaterials described in the third aspect in the production of D-mannose.

[0042] As a fifth aspect of the invention, it is provided to provide the application of the biomaterials described in the third aspect in any of the following aspects:

[0043] (1) Improve the conversion efficiency of D-mannose;

[0044] (2) Inhibit the formation of glucose and fructose byproducts.

[0045] As a sixth aspect of the present invention, a multi-stage enzyme-linked catalytic system is provided to achieve efficient synthesis of D-mannose. The multi-stage enzyme-linked catalytic system comprises: a substrate maltodextrin, α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase described in the first aspect, and mannose-6-phosphate phosphatase; or comprises: a substrate maltodextrin, and genes expressing α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase, and mannose-6-phosphate phosphatase.

[0046] α-glucan phosphorylase originates from Arabidopsis thalianaThe gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase originates from Thermococcus kodakarensis The gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose-6-phosphate isomerase / mannose-6-phosphate isomerase originates from... Dictyoglomus turgid The gene's serial number on Uniprot is B8E0A4; mannose-6-phosphate phosphatase originates from... Pseudothermogo hypogea The gene is numbered A0A0X1KRU1 on Uniprot.

[0047] Furthermore, the multi-level enzyme-linked catalytic system also includes isoamylase, 4-glucantransferase (4-GT), and polyphosphoglucose kinase (PPGK); or expresses the isoamylase gene, 4-glucantransferase gene, and polyphosphoglucose kinase gene.

[0048] The isoamylase is derived from Pseudomonas amyloderamos The gene is numbered P10342 on Uniprot; polyphosphoglucosamine originates from Thermobifida halotolerans The gene's KEGG index is NI17_011080; the 4-glucan transferase gene originates from... Thermococcus siberica The gene is numbered TSIB_0455 on KEGG. These genomic DNA sequences are available from the ATCC official website (www.atcc.org).

[0049] In the multi-stage enzyme-linked catalytic system, maltodextrin is treated with engineered bacteria expressing an isoamylase gene. This enzyme catalyzes the debranching of starch to generate amylose. Then, engineered bacteria expressing α-glucan phosphorylase, glucose phosphate mutase, bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase, and mannose-6-phosphate phosphatase genes convert maltodextrin to D-mannose. In addition, engineered bacteria expressing a 4-glucan transferase gene are added to the expression system. This enzyme catalyzes the polymerization of maltobiose and maltotriose to generate dextrin with a higher degree of polymerization. Engineered bacteria expressing a polyphosphoglucokinase gene can specifically transfer the terminal phosphate group of polyphosphate to glucose molecules to generate glucose-6-phosphate. Glucose-6-phosphate re-enters the D-mannose synthesis pathway, further increasing the D-mannose yield.

[0050] As a seventh aspect of the invention, a recombinant microorganism is provided that expresses the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant described in the first aspect. The microorganism contains the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant or a gene encoding the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant.

[0051] As an eighth aspect of the present invention, there is a method for efficiently synthesizing D-mannose, which uses the biomaterials described in the third aspect, the multi-stage enzyme-linked catalytic system described in the sixth aspect, or the recombinant microorganisms described in the seventh aspect to carry out a catalytic reaction to generate D-mannose.

[0052] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0053] 1. This invention screens key mutation sites through bioinformatics analysis and successfully constructs a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant by combining saturation mutagenesis technology. This mutant can be efficiently expressed in both Escherichia coli and Bacillus subtilis, which not only significantly improves the conversion efficiency of D-mannose, but also effectively inhibits the generation of by-products, providing an efficient and stable biocatalyst for the industrial production of D-mannose.

[0054] 2. This invention, through the analysis of substances derived from... Dictyoglomus turgidum Saturation mutations were performed at single and combined sites on the bifunctional enzyme glucose-phosphorosimide / mannose-phosphorosimide at positions 28 (glutamic acid), 83 (arginine), 126 (valine), 161 (isoleucine), 187 (leucine), 249 (phenylalanine), 319 (leucine), and 343 (tyrosine). Mutants that improved D-mannose conversion efficiency and effectively inhibited the formation of glucose and fructose byproducts were screened. Compared to the wild type, the optimal mutant increased the D-mannose yield to 95.05% while effectively inhibiting byproduct formation, reducing the proportions of glucose and fructose byproducts by 55.87% and 58.10%, respectively. This invention provides a highly efficient and stable biocatalyst for the industrial production of D-mannose and has significant application value.

[0055] 3. As a GRAS (Generally Regarded As Safe) strain, *Bacillus subtilis* does not produce pyrogens or endotoxins and has no toxic side effects, which can reduce the cost of subsequent separation and purification, making it suitable for the production of D-mannose. Currently, there are few techniques or reports on the de novo synthesis of D-mannose using *Bacillus subtilis*. The mutant constructed in this invention also exhibits excellent catalytic performance in the *Bacillus subtilis* expression system. Compared to the wild type, the optimal mutant significantly increases the D-mannose yield to 88.42%. Therefore, this invention has high application value. Attached Figure Description

[0056] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0057] Figure 1 This is a process flow diagram of the multi-stage enzyme-linked catalytic conversion of D-mannose using maltodextrin as a substrate. Detailed Implementation

[0058] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0059] The present invention will be further described below with reference to the embodiments.

[0060] In some embodiments of the present invention, a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant is provided, wherein the amino acid sequence of the mutant is mutated at at least one of the following positions: glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0061] In some embodiments of the present invention, the amino acid point mutations of the mutant, compared to the wild-type enzyme with an amino acid sequence as shown in SEQ ID NO.1, are selected from at least one of the following:

[0062] (1) The glutamic acid at position 28 is mutated to serine or aspartic acid;

[0063] (2) The arginine at position 83 is mutated to lysine or tryptophan;

[0064] (3) The valine at position 126 is mutated to isoleucine or phenylalanine;

[0065] (4) The isoleucine at position 161 is mutated to tryptophan or valine;

[0066] (5) The leucine at position 187 is mutated to methionine or isoleucine;

[0067] (6) The phenylalanine at position 249 is mutated to isoleucine or tyrosine;

[0068] (7) The leucine at position 319 is mutated to phenylalanine or tryptophan;

[0069] (8) The tyrosine at position 343 is mutated to phenylalanine or methionine.

[0070] In some embodiments of the present invention, a gene encoding the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant is provided.

[0071] In some embodiments of the present invention, biological materials comprising a gene encoding a mutant of the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase are provided, the biological materials comprising an expression cassette, a vector, or a host cell; the host cell being selected from Escherichia coli and Bacillus subtilis.

[0072] In some embodiments of the present invention, the application of the biomaterials described herein is provided in the production of D-mannose.

[0073] In some embodiments of the present invention, the biomaterial has the following functions in the production of D-mannose:

[0074] (1) Improve the conversion efficiency of D-mannose;

[0075] (2) Inhibit the formation of glucose and fructose byproducts.

[0076] In some embodiments of the present invention, a multi-stage enzyme-linked catalytic system is provided to achieve efficient synthesis of D-mannose. The multi-stage enzyme-linked catalytic system comprises: substrate maltodextrin, α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase described in the first aspect, and mannose-6-phosphate phosphatase; or comprises: substrate maltodextrin, and genes expressing α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzymes glucose phosphate isomerase / mannose phosphate isomerase, and mannose-6-phosphate phosphatase.

[0077] Furthermore, the multi-level enzyme-linked catalytic system also includes isoamylase, 4-glucantransferase, and polyphosphoglucose kinase; or engineered bacteria expressing isoamylase genes, 4-glucantransferase genes, and polyphosphoglucose kinase genes.

[0078] In some embodiments of the present invention, a recombinant microorganism is provided that expresses the aforementioned bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant. The microorganism contains the mutant or a gene encoding the mutant.

[0079] In some embodiments of the present invention, a method for efficiently synthesizing D-mannose is provided, using the aforementioned biomaterials, or the multi-stage enzyme-linked catalytic system described in the sixth aspect, or the recombinant microorganisms described in the seventh aspect, to catalyze a reaction to generate D-mannose. A process flow diagram of the multi-stage enzyme-linked catalytic conversion of D-mannose using maltodextrin as a substrate is shown below. Figure 1 As shown.

[0080] In some embodiments, the concentration of isoamylase in the reaction system is 0.1-2 wt%, preferably 0.4 wt%; the concentration of 4-glucan transferase gene is 0.1-3 wt%, and in some embodiments, the concentration is any value between 0.5 wt%, 1 wt%, 1.5 wt%, 2.5 wt%, or 0.5-2.5 wt%, preferably 1 wt%; the concentration of polyphosphoglucose kinase is any value between 0.1-2 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, or 0.5-1.5 wt%, preferably 1.5 wt%; and the concentration of α-glucan phosphorylase is 0.5-5 wt%, or 1 wt%, 2 wt%, 3 wt%, or 0.5 wt%. The concentration of glucose phosphate isomerase is 0.5-5 wt% or any value between 1 wt%, 1.5 wt%, 2 wt%, 3 wt% or 1-3 wt%, preferably 1 wt%; the concentration of the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant is 0.5-5 wt% or any value between 1 wt%, 1.5 wt%, 2 wt%, 3 wt% or 1-3 wt%, preferably 2 wt%; the concentration of mannose 6-phosphate phosphatase is 2-10 wt% or any value between 2 wt%, 3 wt%, 4 wt%, 6 wt% or 2-6 wt%, preferably 4 wt%.

[0081] In one or more embodiments, the temperature for treating maltodextrin with engineered bacteria expressing the isoamylase gene is 70–85°C, preferably 80°C; the treatment time is 8–16 h, preferably 12 h. The concentration of maltodextrin is 1–500 g / L, preferably 150 g / L; the concentration of phosphate is 1–100 mM, preferably 10 mM; Mg 2+ The concentration of the active ingredient is 1~50 mM, preferably 5 mM; the final concentration of sodium hexametaphosphate is 30~70 mM, preferably 40 mM. The reaction temperature is between 40~80℃, preferably 65℃; the reaction time is between 8~48h, preferably 24h.

[0082] The KPB phosphate buffer solution used in this embodiment is prepared by using potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) as solutes, with a pH of 6.8~7.2, and the concentrations of potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) are both 10 mM or 20 mM.

[0083] Example 1: Construction and expression analysis of PGI / PMI vector in Escherichia coli

[0084] Includes the following steps:

[0085] (a) will originate from Dictyoglomus turgidumThe amino acid sequence of the glucose-phosphoryl isomerase / mannose-phosphoryl isomerase gene fragment (PGI / PMI) of the genus *P.* was codon-optimized and recombined into the pET32a vector to obtain the recombinant plasmid pET32a-PGI / PMI. Its corresponding amino acid sequence is shown in SEQ ID NO.1.

[0086] (b) The recombinant plasmid pET32a-PGI / PMI was transformed into the Escherichia coli expression host BL21(DE3) to obtain the prokaryotic expression strain.

[0087] (c) The recombinant strain containing the recombinant plasmid pET32a-PGI / PMI was inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and activated overnight to obtain seed culture. The seed culture was then transferred to 100 mL of LB medium (containing 100 μg / mL ampicillin) at a 1% inoculation rate and cultured until OD. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM to induce protein expression. Collect bacterial cells by centrifugation, weigh out bacterial sludge at a ratio of 20 wt%, and suspend the cells in 20 mM KPB phosphate buffer (pH=7.0). After ultrasonic disruption, run a protein gel to verify the expression effect of the pET32a-PGI / PMI gene. The PGI / PMI protein band size was approximately 58.96 kDa.

[0088] (d) The PGI / PMI enzyme activity assay system is as follows: In a 1 mL reaction system, add 50 mM glucose-6-phosphate, 10 mM KPB phosphate buffer (pH=7.0), 5 mM MgCl2, 5 U / mL mannose-6-phosphate phosphatase, and 10~50 u LGI / PMI crude enzyme solution (the amount of enzyme solution added is adjusted according to the measured D-mannose production, with the appropriate amount of enzyme required to produce 5 mM D-mannose as the optimal amount). React at 65℃ for 10~20 min. After the reaction, boil for 5 min, centrifuge to remove impurities, and detect the D-mannose production using HPLC to calculate the enzyme activity. The HPLC analysis conditions are as follows: Thermo Scientific™ high-performance liquid chromatograph, Shodex SUGAR column, column temperature: 80℃, mobile phase: ddH2O, flow rate: 0.6 mL / min, injection volume: 20 μL.

[0089] Example 2, Construction of the PGI / PMI mutant library

[0090] Includes the following steps:

[0091] (a) Bioinformatics analysis of the amino acid sequence and structure of wild-type PGI / PMI was performed to identify key amino acid residues in the enzyme's catalytic activity center region. The amino acid residues near the substrate channel were also analyzed, and eight sites that may affect catalytic activity and substrate-specific binding ability were finally screened out: glutamic acid at position 28, arginine at position 83, valine at position 126, isoleucine at position 161, leucine at position 187, phenylalanine at position 249, leucine at position 319, and tyrosine at position 343.

[0092] (b) Single-point saturation mutations were performed at positions 28, 83, 126, 161, 187, 249, 319, and 343, respectively, to design mutation primers for the specified mutation sites. Using the recombinant plasmid pET32a-PGI / PMI provided in Example 1 as a template, the recombinant plasmid PGI / PMI was amplified by full plasmid PCR in the presence of high-fidelity Pha enzyme using each mutation primer to obtain linearized plasmids at the specified mutation sites. The amplification products were digested with DpnI enzyme at 37°C for 1 h, and then transformed into Escherichia coli BL21(DE3) competent cells, plated on LB plates (containing 100 μg / mL ampicillin), and cultured overnight at 37°C to obtain positive clone transformants.

[0093] (c) Obtaining crude enzyme solution from mutants: Strains from each mutant, after sequencing verification of the corresponding mutation sites, were subjected to activity testing; positive clone transformants were inoculated at a rate of 1% into 500 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37℃ and 220 rpm until OD500. 600 At a concentration of 0.6–0.8, IPTG was added to a final concentration of 0.1 mM, and the cells were collected by centrifugation after induction at 25°C for 16 h. The bacterial sludge was weighed at a ratio of 20 wt% and resuspended in 20 mM KPB phosphate buffer. The resuspended bacterial sludge was then disrupted using an ultrasonic homogenizer to obtain crude enzyme solution.

[0094] Example 3: Catalytic specificity analysis of PGI / PMI unit point mutants using maltodextrin as a substrate

[0095] Includes the following steps:

[0096] (a) Based on the crude enzyme solution described in Example 2 above, a mutation site screening system was established: 1 mL of the reaction system contained 150 g / L maltodextrin treated with 0.4 wt% IA and 5 mM Mg 2+The enzyme solution was prepared with 10 mM KPB phosphate buffer (pH=7.0), and crude enzyme solutions consisting of αGP, PGM, PGI / PMI, and M6PP were added to achieve enzyme concentrations of 1 wt%, 1 wt%, 2 wt%, and 4 wt%, respectively. The solution was then brought to a final volume of 1 mL with ddH2O. The reaction was carried out in a 65℃ constant-temperature reactor at 700 rpm for 24 h. Samples were then collected. Sample processing: After centrifugation, the supernatant was diluted 10-fold, filtered through a 0.22 μm filter, and analyzed by HPLC. The yields of D-mannose, glucose, and fructose were determined using high-performance liquid chromatography.

[0097] (b) In one or more embodiments, the isoamylase is derived from Pseudomonas amyloderamos The gene is numbered P10342 on Uniprot; α-glucan phosphorylase originates from Arabidopsis thaliana The gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase originates from Thermococcus kodakarensis The gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose-6-phosphate isomerase / mannose-6-phosphate isomerase originates from... Dictyoglomus turgidum The gene's serial number on Uniprot is B8E0A4; mannose-6-phosphate phosphatase originates from... Pseudothermogo hypogea The gene is numbered A0A0X1KRU1 on Uniprot.

[0098] (c) Mutant Validation and Characterization: The specificity-enhancing mutants obtained from the initial screening were sequenced and identified to determine the key amino acid substitution types. Mutants exhibiting both high specificity and high catalytic activity were screened, and the results are shown in Table 1.

[0099] Table 1. Catalytic specificity and catalytic activity analysis of PGI / PMI unit point mutants using maltodextrin as a substrate.

[0100]

[0101] Table 1 shows that amino acid residues at positions 28, 83, 126, 161, 187, 249, 319, and 343 play a crucial regulatory role in the enzyme's catalytic activity and substrate specificity. In a multi-enzyme cascade reaction system using maltodextrin as a substrate, the introduction of a single-point-mutated glucose-phosphoisomerase / mannose-phosphoisomerase bifunctional mutant significantly improved the catalytic efficiency compared to the wild type: the optimal mutant's mannose conversion rate increased from 72.36% in the control group to 83.62%.

[0102] Example 4: Analysis of catalytic specificity and catalytic activity of the PGI / PMI combined site mutant using maltodextrin as a substrate.

[0103] The above sites were combined with saturation mutations, and the catalytic specificity and catalytic activity of the PGI / PMI combined site mutants were evaluated using the method described in Example 3. The results are shown in Table 2.

[0104] Table 2. Catalytic specificity and activity analysis of PGI / PMI combined site mutants using maltodextrin as a substrate.

[0105]

[0106] Table 2 shows that in a multi-enzyme cascade reaction system using maltodextrin as a substrate, introducing bifunctional glucose-phosphoisomerase / mannose-phosphoisomerase mutants with combined site mutations at amino acids 28, 83, 126, 161, 187, 249, 319, and 343 significantly improved catalytic efficiency compared to the wild type: the optimal mutant increased mannose conversion from 72.36% in the control group to 90.50%, and reduced the proportions of glucose and fructose byproducts to 55.87% and 58.10%, respectively. This confirms the optimizing effect of key site mutations on enzyme function.

[0107] Example 5: Optimization of enzyme ratios in the multi-enzyme reaction system for the synthesis of D-mannose:

[0108] (a) A 1 mL reaction system contains 150 g / L maltodextrin treated with 0.4 wt% IA and 5 mM Mg 2+ Add 10 mM phosphate buffer (pH=7.0), and then add crude enzyme solutions composed of αGP, PGM, PGI / PMI (mutant 34, hereinafter referred to as M34), and M6PP, respectively. Adjust the volume to 1 mL with ddH2O. Incubate in a 65℃ constant-temperature reactor at 700 rpm for 24 h, then sample. Sample processing: Centrifuge the finished sample, dilute the supernatant 10-fold, filter through a 0.22 μm filter membrane, and perform HPLC analysis.

[0109] Table 3. Reaction results with different enzyme ratios

[0110]

[0111] Table 3 shows that the highest D-mannose production rate was 89.76% when the addition amounts of aGP were 1 wt%, PGM were 1 wt%, PGI / PMI (M34) were 2 wt%, and M6PP were 4 wt%.

[0112] (b) To further improve the conversion rate, 4-glucan transferase and polyphosphoglucoskinase were added to the reaction system. Each 1 mL reaction system contained 150 g / L maltodextrin treated with 0.4 wt% IA and 5 mM Mg...2+ Add crude enzyme solutions composed of αGP, PGM, PGI / PMI, and M6PP to 10 mM phosphate buffer (pH=7.0) to achieve enzyme concentrations of 1 wt%, 1 wt%, 2 wt%, and 4 wt%, respectively. Adjust the volume to 1 mL with ddH2O. Incubate the reaction in a 65℃ constant-temperature reactor at 700 rpm for 24 h. Afterward, take 500 μL of the mannose reaction solution, add phosphate buffer to a final concentration of 10 mM, and then add Mg... 2+ The final concentration of the reagent was 5 mM, sodium hexametaphosphate 40 mM, and crude enzyme solutions of 4-glucan transferase and polyphosphoglucoskinase were added. Water was added to bring the volume to 1 mL. The mixture was placed in a 65℃ constant temperature reactor and reacted at 700 rpm for 8 h. Samples were then taken. Sample preparation: After centrifugation, the supernatant of the reacted sample was diluted 10-fold, filtered through a 0.22 μm filter membrane, and analyzed by HPLC.

[0113] Table 4. Reaction results with different enzyme ratios

[0114]

[0115] Table 4 shows that when the addition amount of 4-GT is 1 wt% and the addition amount of PPGK is 1.5 wt%, the conversion rate of D-mannose can reach up to 97.08%.

[0116] Example 6: Whole-cell catalytic production of D-mannose using Bacillus subtilis as the expression host.

[0117] Includes the following steps:

[0118] (a) Construction of engineered bacteria expressing α-glucan phosphorylase gene, glucose phosphate mutase gene, bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase (M34) gene and mannose-6-phosphate phosphatase gene selected above with high catalytic specificity and / or activity in Bacillus subtilis.

[0119] In this embodiment, α-glucan phosphorylase is derived from Arabidopsis thaliana The gene is numbered Q9SD76 on Uniprot; glucose-phosphoryltransferase originates from Thermococcus kodakarensis The gene's Uniprot number is Q68BJ6; the bifunctional enzyme glucose phosphate isomerase / mannose 6-phosphate isomerase mutant is derived from the invention described above; mannose 6-phosphate phosphatase is derived from... Pseudothermogo hypogea The gene is numbered A0A0X1KRU1 on Uniprot.

[0120] The specific steps are as follows: Using PWB980 as the vector, corresponding primers for each gene were designed to amplify the vector and fragments. The amplified fragments were then ligated into SCK6 competent cells via PCR, plated on LB agar plates containing kanamycin (25 μg / mL), and cultured overnight. Transformants were selected for testing. The successfully constructed strains were named PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI(M34), and PWB980-M6PP, respectively.

[0121] (b) Using engineered bacteria expressing the α-glucan phosphorylase gene, glucose phosphate mutase gene, a bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant gene with high catalytic specificity and / or activity, and mannose-6-phosphate phosphatase gene, an enzyme for the whole-cell catalytic preparation of D-mannose was obtained. The specific steps are as follows: The PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI (M34), and PWB980-M6PP strains constructed in step (a) were inoculated into test tubes containing 5 mL of LB liquid medium with kanamycin resistance (50 μg / mL) and cultured at 37°C and 200 rpm for 16 h in a constant temperature shaker. After culturing, the seed culture was transferred at a 2% inoculation rate to a shake flask containing 400 mL of LB liquid medium with kanamycin resistance (50 μg / mL). The culture was incubated at 37°C and 200 rpm for 48 h in a shaker. The cells were collected by centrifugation at 8000 rpm for 15 min. A 20 wt% sample of bacterial sludge was weighed and resuspended. The resuspended sludge was then ultrasonically broken up to obtain a crude enzyme solution.

[0122] (c) Based on the crude enzyme solution obtained above, a reaction system was established: 1 mL of the reaction system contained 150 g / L maltodextrin treated with 0.4% IA and 5 mM Mg 2+ The mixture was prepared with 10 mM phosphate buffer (pH=7.0), and crude enzyme solutions consisting of PWB980-αGP, PWB980-PGM, PWB980-PGI / PMI (M34), and PWB980-M6PP were added at concentrations of 2 wt%, 1 wt%, 1 wt%, and 4 wt%, respectively. The concentrations of 4GT were 1 wt%, and the concentration of PPGK was 1.5 wt%. The volume was adjusted to 1 mL with ddH2O. The reaction was carried out in a 65℃ constant-temperature reactor at 700 rpm for 24 h. Samples were taken after the reaction and analyzed by HPLC. Using D-mannose conversion rate as the evaluation index of catalytic specificity, the D-mannose conversion rate obtained in this example reached 88.42%.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant, characterized in that, Compared to the wild-type enzyme with an amino acid sequence as shown in SEQ ID NO.1, the mutant is selected from any of the following: (1) Glutamic acid at position 28 is mutated to serine, and arginine at position 83 is mutated to lysine; (2) Glutamic acid at position 28 is mutated to serine, and arginine at position 83 is mutated to tryptophan; (3) Glutamic acid at position 28 is mutated to serine, and valine at position 126 is mutated to isoleucine; (4) Glutamic acid at position 28 is mutated to serine, and isoleucine at position 161 is mutated to tryptophan; (5) Glutamic acid at position 28 is mutated to serine, and leucine at position 187 is mutated to methionine. (6) Glutamic acid at position 28 is mutated to serine, and phenylalanine at position 249 is mutated to isoleucine; (7) Glutamic acid at position 28 is mutated to serine, and phenylalanine at position 249 is mutated to tyrosine; (8) Glutamic acid at position 28 is mutated to serine, while leucine at position 319 is mutated to phenylalanine and tyrosine at position 343 is mutated to methionine. (9) Glutamic acid at position 28 is mutated to serine, while leucine at position 319 is mutated to tryptophan and tyrosine at position 343 is mutated to phenylalanine. (10) Glutamic acid at position 28 is mutated to serine, while arginine at position 83 is mutated to lysine and tyrosine at position 343 is mutated to methionine. (11) Glutamic acid at position 28 is mutated to serine, while arginine A at position 83 is mutated to lysine, valine at position 126 is mutated to phenylalanine, and isoleucine at position 161 is mutated to valine. (12) Glutamic acid at position 28 is mutated to serine, while arginine A at position 83 is mutated to lysine, valine at position 126 is mutated to phenylalanine, and tyrosine at position 343 is mutated to methionine. (13) Glutamic acid at position 28 is mutated to serine, while valine at position 126 is mutated to phenylalanine, isoleucine at position 161 is mutated to valine, leucine at position 187 is mutated to methionine, and phenylalanine at position 249 is mutated to tyrosine. (14) Glutamic acid at position 28 is mutated to serine, while arginine at position 83 is mutated to lysine, valine at position 126 is mutated to phenylalanine, isoleucine at position 161 is mutated to valine, leucine at position 187 is mutated to methionine, phenylalanine at position 249 is mutated to tyrosine, leucine at position 319 is mutated to phenylalanine, and tyrosine at position 343 is mutated to methionine.

2. The gene encoding the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of claim 1.

3. A biological material comprising the gene of claim 2, wherein the biological material comprises an expression cassette, a vector, or a host cell.

4. The application of the biomaterial described in claim 3 in the production of D-mannose.

5. The application of the biomaterial according to claim 4 in the production of D-mannose, characterized in that, (1) Improve the conversion efficiency of D-mannose; (2) Inhibit the formation of glucose and fructose byproducts.

6. A multi-stage enzyme-linked catalytic system, characterized in that, Contains: substrate maltodextrin, α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of claim 1, and mannose-6-phosphate phosphatase; or contains: substrate maltodextrin, and genes expressing α-glucan phosphorylase, glucose phosphate mutase, the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of claim 1, and mannose-6-phosphate phosphatase. The multi-level enzyme-linked catalytic system also includes engineered bacteria expressing isoamylase genes, 4-glucan transferase genes, and polyphosphoglucose kinase genes.

7. A recombinant microorganism, characterized in that, The microorganism contains the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of claim 1 or the gene encoding the bifunctional enzyme glucose phosphate isomerase / mannose phosphate isomerase mutant of claim 1.

8. A method for synthesizing D-mannose, characterized in that, The biomaterials of claim 3, the multi-stage enzyme-linked catalytic system of claim 6, or the recombinant microorganisms of claim 7 are used to catalyze a reaction to generate D-mannose.

Images