Heat-resistant trehalose-6-phosphate phosphatase mutant and application thereof

Abstract

The application relates to a heat-resistant trehalose-6-phosphate phosphatase mutant and application thereof, and belongs to the technical field of genetic engineering and enzyme engineering. The mutant is obtained by single-point mutation or combined mutation on the basis of the amino acid sequence shown in SEQ ID NO: 2, and the mutation sites include E100M, A106R, T235V, L580Y, T510M, E595A, A702V, N602P, G430S, E595P and S607A. The heat stability of the mutant is higher than that of the wild type, and the half-life of the combined mutant E100M / T235V / T510M / G430S / A702V / E595P / S607A at 60 DEG C reaches 17.3 h. The mutant can be used for constructing a catalytic system together with maltose phosphorylase and polyphosphate-dependent glucose kinase, and for producing trehalose-6-phosphate by taking maltose and polyphosphate as substrates.

Description

Technical Field

[0001] This application relates to a heat-resistant trehalose-6-phosphate phosphorylase mutant and its application, belonging to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] Trehalose-6-phosphate (T6P) is an important signaling molecule in plants. During plant growth and development, T6P mainly participates in starch accumulation, growth and development, branching and flowering by inhibiting sucrose non-fermentation 1-related protein kinase (SnRK1). It also mediates responses to abiotic stresses such as drought and low temperature. As a sugar signaling biostimulant, T6P has the ability to increase crop yield and improve quality. Developing efficient T6P synthesis technology is an important requirement for promoting the improvement of agricultural production quality and efficiency.

[0003] Enzymatic synthesis of T6P offers advantages such as mild reaction conditions and high product yield. Trehalose-6-phosphate synthase (TPS) can catalyze the synthesis of T6P from glucose-6-phosphate (G6P) and UDP-glucose (UDPG), but it suffers from bottlenecks such as the high cost of the substrate UDPG, hindering industrialization. Trehalose-6-phosphate phosphorylase (TPP, EC 2.4.1.216) synthesizes T6P using G6P and β-glucose-1-phosphate (βG1P) as substrates, offering a substrate cost advantage over TPS-catalyzed T6P synthesis. As early as 2001, Ulrika Andersson et al. reported on the synthesis of T6P from Lactococcus lactis (…). Lactococcus lactis TPP derived from sodium hexametaphosphate has the ability to catalyze the formation of T6P from substrates. Based on this, in 2026, Liu et al. constructed a three-enzyme catalytic system using maltose phosphorylase MP purified by nickel column, polyphosphate-dependent glucokinase PPGK, and TPP, and successfully synthesized T6P using maltose and sodium hexametaphosphate as substrates.

[0004] Microorganisms contain a large number of dephosphorylases, which can lead to the dephosphorylation of the product T6P to generate trehalose, thus reducing the conversion rate. When enzyme elements such as MP, PPGK, and TPP from room-temperature bacteria are heterologously expressed in E. coli, the existing three-enzyme catalytic system requires nickel column purification of the enzyme elements to remove the endogenous dephosphorylases from E. coli, resulting in high enzyme usage costs and limiting their industrial application.

[0005] Therefore, it is urgent to create thermophilic MP, PPGK, and TPP by using enzyme element mining and enzyme engineering techniques, and to reduce the cost of enzyme use by inactivating endogenous dephosphorases of E. coli through one-step heat treatment, thereby further promoting the industrialization of T6P production using the three-enzyme system. Summary of the Invention

[0006] To address the aforementioned issues, a heat-resistant trehalose-6-phosphate phosphorylase mutant and its applications are provided. This mutant exhibits superior heat resistance and can synergistically catalyze with maltose phosphorylase and polyphosphate-dependent glucokinase to efficiently synthesize trehalose-6-phosphate under high-temperature conditions using maltose and polyphosphate as substrates, thereby improving substrate conversion.

[0007] This application provides a thermostable trehalose-6-phosphate phosphorylase mutant, the amino acid sequence of which is obtained by substituting any one of the following amino acids based on the amino acid sequence shown in SEQ ID NO:2: 1) E100M; 2) A106R; 3) T235V; 4) E100M / T235V; 5) E100M / T235V / L580Y; 6) E100M / T235V / T510M; 7) E100M / T235V / T510M / E595A / A702V; 8) E100M / T235V / T510M / E595A / N602P; 9) E100M / T235V / T510M / G430S / A702V; 10) E100M / T235V / T510M / E595P / S607A; 11) E100M / T235V / T510M / G430S / A702V / E595A; 12) E100M / T235V / T510M / G430S / A702V / E595P / S607A; The positions of the amino acid residues are numbered based on the amino acid sequence shown in SEQ ID NO:2, and the slashes indicate that the listed amino acid substitutions are present simultaneously.

[0008] Optionally, the amino acid sequence of the mutant is shown in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.

[0009] This application provides a method for preparing the above-mentioned trehalose-6-phosphate phosphorylase mutant, wherein a vector containing the gene encoding the trehalose-6-phosphate phosphorylase mutant is introduced into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria are cultured to express the trehalose-6-phosphate phosphorylase mutant.

[0010] Optionally, after culturing the recombinant bacteria, the collected recombinant bacteria may be treated with any of the following methods to obtain a formulation containing the trehalose-6-phosphate phosphorylase mutant: 1) The recombinant bacteria were subjected to heat treatment to obtain a whole-cell catalyst; 2) After cleaving the recombinant bacteria, a crude enzyme solution was obtained. The crude enzyme solution was purified by nickel column chromatography to obtain the trehalose-6-phosphate phosphorylase mutant.

[0011] This application provides a biomaterial, which is any one of the following: 1) A nucleic acid molecule encoding the aforementioned trehalose-6-phosphate phosphorylase mutant; 2) An expression cassette, the expression cassette comprising the nucleic acid molecule described in item 1) and a promoter operatively linked to the nucleic acid molecule; 3) A recombinant vector comprising the nucleic acid molecule described in item 1) or the expression cassette described in item 2); 4) Recombinant cells, said recombinant cells comprising the nucleic acid molecule described in item 1), the expression cassette described in item 2), or the recombinant vector described in item 3); 5) Whole-cell catalyst, wherein the whole-cell catalyst is prepared by collecting and heat treatment after the recombinant cells described in item 4) express the trehalose-6-phosphate phosphorylase mutant.

[0012] This application provides a method for preparing trehalose-6-phosphate, using maltose and polyphosphate as substrates, and utilizing maltose phosphorylase, polyphosphate-dependent glucokinase and the above-mentioned trehalose-6-phosphate phosphorylase mutant to carry out a catalytic reaction to generate trehalose-6-phosphate. The maltose phosphorylase catalyzes the reaction of maltose with inorganic phosphate to produce glucose and β-glucose-1-phosphate, the polyphosphate-dependent glucokinase utilizes polyphosphate as a phosphate donor to catalyze the reaction of glucose with glucose-6-phosphate, and the trehalose-6-phosphate phosphorylase mutant catalyzes the reaction of β-glucose-1-phosphate with glucose-6-phosphate to produce trehalose-6-phosphate.

[0013] Optionally, the maltose phosphorylase is *Thermophilus xivans* (…). Thermophagus xiamenensis (Source) and / or, The polyphosphate-dependent glucokinase is *Pseudomonas thermophila* (…). Pseudonocardia thermophila (Source)

[0014] Optionally, the catalytic reaction employs a whole-cell catalyst, which is prepared using *E. coli* as a host and through any of the following methods: 1) Construct three recombinant cells expressing one of the three enzymes respectively, culture and collect each recombinant cell, perform heat treatment separately and then combine them to obtain the whole-cell catalyst; 2) Construct recombinant cells that co-express the three enzymes in the same cell, culture and collect the recombinant cells, and obtain the whole-cell catalyst by heat treatment; 3) Construct a first recombinant cell that co-expresses any two of the three enzymes in the same cell, and a second recombinant cell that expresses the remaining enzyme. Culture and collect the first recombinant cell and the second recombinant cell respectively, perform heat treatment on each cell, and then combine them to obtain the whole-cell catalyst. The three enzymes are maltose phosphorylase, polyphosphate-dependent glucokinase, and trehalose-6-phosphate phosphorylase mutant.

[0015] Optionally, a catalytic reaction can be carried out at 50~60℃ to generate trehalose-6-phosphate.

[0016] Optionally, the catalytic system for preparing trehalose-6-phosphate may also include magnesium chloride and / or magnesium sulfate.

[0017] The beneficial effects of this application include, but are not limited to: Compared with existing technologies, the trehalose-6-phosphate phosphorylase mutant provided in this application exhibits improved thermal stability. Specifically, the E100M / T235V / T510M / G430S / A702V / E595P / S607A combination mutant has a half-life of 17.3 h at 60°C, and its specific activity is 1.7 times that of the wild type. When the mutant is used in combination with thermostable maltose phosphorylase and polyphosphate-dependent glucokinase, trehalose-6-phosphate can be synthesized efficiently at 55°C using maltose and polyphosphate as substrates, achieving a maltose molar conversion rate of 94.3%.

[0018] Furthermore, whole-cell catalysts can be prepared by heat-treating recombinant cells expressing the corresponding enzymes, reducing product loss caused by endogenous dephosphorylases in the host and improving cell permeability. This eliminates the need for enzyme separation and purification, which helps reduce catalyst preparation costs and enables large-scale production of trehalose-6-phosphate. Attached Figure Description

[0019] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This invention relates to the thermal stability of wild-type trehalose-6-phosphate phosphorylase and its mutants at 60°C in the embodiments of this application.

[0020] Figure 2The image shows the reaction process curve of trehalose-6-phosphate synthesized from maltose and sodium hexametaphosphate at 55°C using a three-enzyme catalysis method, as described in the embodiments of this application. Detailed Implementation

[0021] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0022] The method for constructing the TPP mutant vector in this application is as follows: Using the Phusion site-directed mutagenesis method, conventional primers were designed, and PCR amplification was performed using either the vector pET28a-tpp containing wild-type TPP or a vector containing the optimal mutant of each round as a template. The PCR reaction system was: 2... 25 μL of PrimeSTAR mixture, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, and 22 μL of double-distilled water were added. PCR amplification conditions were as follows: 98℃ pre-denaturation for 45 s; followed by 30 cycles of 98℃ for 15 s, 55℃ for 15 s, and 72℃ for 1 min, with a final incubation at 72℃ for 5 min. The PCR product was digested with Dpn I for 30 min and then purified using a PCR product purification kit. 3 μL of the purified PCR product was mixed with 1 μL of T4PNK and 10 μL of... Mix 1 μL of T4 PNK buffer, 1 μL of 100 mM ATP, and 4 μL of double-distilled water, and incubate at 37°C for 2 h for phosphorylation. Take 10 μL of the phosphorylated PCR product solution, mix with 1 μL of rapid ligase and 10 μL of rapid ligase buffer, and incubate at 25°C for 30 min for blunt-end ligation. Transform the ligation products into competent *E. coli* TOP10 cells, plate them onto LB agar plates containing antibiotics, and incubate overnight. Pick single colonies, incubate overnight in LB liquid medium, extract plasmids, and perform sequencing verification.

[0023] The expression method of the TPP mutant in this application is as follows: BL21(DE3) strain containing the correctly sequenced mutant gene vector was selected and inoculated into LB liquid medium. The culture was carried out at 37℃ and 200 rpm until the OD600nm reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM for induction, and the culture was continued at 25℃ for 12 h. The bacterial cells were collected, and the crude mutant protein enzyme solution was obtained by ultrasonic disruption. The solution was purified by nickel column chromatography, concentrated by ultrafiltration, and the buffer was replaced to obtain electrophoretically pure mutant protein. The nickel column purification was used to determine the specific enzyme activity and thermal stability.

[0024] The method for determining the specific enzyme activity of TPP in this application is as follows: In a 100 mM HEPES buffer (pH 7.5) containing 10 mM magnesium chloride, β-glucose-1-phosphate (βG1P) and 10 mM glucose-6-phosphate (G6P) are added to a final concentration of 10 mM. The mixture is reacted at 55°C for 10 min, and the amount of inorganic phosphorus produced is measured.

[0025] The method for determining the thermal stability of TPP in this application is as follows: Wild-type or mutant protein is diluted to a final concentration of 1 g / L (100 mM HEPES, pH 7.5), placed in a 60°C water bath, and samples are taken at different times to determine residual enzyme activity. The specific enzyme activity of the protein after 0 h of incubation is taken as 100%.

[0026] The present application solution will be described below through specific embodiments.

[0027] Example 1: First round of site-directed mutagenesis and combined mutagenesis Homology modeling of wild-type TPP (Uniprot ID A0A1D3DN37) from *Streptomyces thermopurpureus* was performed using AlphaFold. FireProt, combined with FoldX energy calculations and evolutionary conservation analysis, was used to screen for energy-high-risk and combined-high-risk regions. Based on folding free energy and conservation scores, six single-point mutation sites (V72M, E100M, A106R, A217M, T235V, C678M) were identified. Using the vector pET28a-tpp containing the codon-optimized wild-type TPP coding gene (as shown in SEQ ID NO:1) as a template, PCR amplification was performed to construct the expression vector for the TPP mutant. The mutant protein was obtained through protein expression, and its thermostability was determined. The half-life of wild-type TPP (as shown in SEQ ID NO:2) at 60°C was 14.9 min. This round of mutations yielded three mutants with improved thermal stability after point mutation: TPP-E100M, TPP-A106R, and TPP-T235V, with half-lives of 33.6 min, 26.2 min, and 43.6 min at 60 °C, respectively.

[0028] Combinatorial mutations were performed on the above sites to obtain the combined mutant TPP-E100M / T235V, which had an increased half-life of 1.78 h at 60℃. The optimal mutant TPP-E100M / T235V obtained in this round has the coding nucleic acid sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:4, and is named R1.

[0029] Example 2: Second round of site-directed mutagenesis and combined mutagenesis Starting with R1 as the starting protein, the stability hotspot module of HotSpot Wizard was used to screen non-catalytic residues in the flexible loop region based on homologous sequence amino acid frequency and mutation landscape analysis, resulting in five single-point thermostability modification candidate sites: D467S, T510M, L580Y, H690Q, and H633Y. Mutant proteins were obtained through protein expression, and their thermostability was measured. This round of mutations yielded two mutants with improved thermostability after point mutation: R1-L580Y and R1-T510M, with half-lives of 3.7 h and 3.96 h at 60 °C, respectively. Combined mutations at the above sites did not yield mutants with improved stability. The optimal mutant obtained in this round, R1-T510M, has the encoding nucleic acid sequence shown in SEQ ID NO:5 and the amino acid sequence shown in SEQ ID NO:6, and is named R2.

[0030] Example 3: Third round of site-directed mutagenesis and combined mutagenesis Starting with R2 as the starting protein, the HotSpot Wizard covariation module was used. Based on the principle of evolutionary covariation, consensus scores, mutagenicity, and key residue characteristics were combined to screen sites, excluding catalytic pockets, to obtain residue pairs suitable for multi-point co-mutation (G332A / P334G, E595A / A702V, E595A / N602P, G430S / A702V, G430S / E595A, W78A / L79Q, E595P / S607A, R19Q / L79Q). The mutant protein was obtained through protein expression, and its thermostability was determined. This round of mutations yielded four mutants with improved thermal stability after point mutation: R2-E595A / A702V, R2-E595A / N602P, R2-G430S / A702V, and R2-E595P / S607A, with half-lives of 6.5 h, 5.6 h, 9.9 h, and 8.9 h at 60 °C, respectively. Combinatorial mutations were then performed on these sites to obtain the combined mutants R2-G430S / A702V / E595A and R2-G430S / A702V / E595P / S607A, with half-lives of 13.4 h and 17.3 h at 60 °C, respectively. The optimal mutant obtained in this round, R2-G430S / A702V / E595P / S607A, has the coding nucleic acid sequence shown in SEQ ID NO:7 and the amino acid sequence shown in SEQ ID NO:8, and is named R3.

[0031] After three rounds of site-directed and combined mutagenesis, the optimal mutant R3 had a half-life of 17.3 h at 60 °C, a 70-fold increase compared to the wild-type half-life of 14.9 min. The thermal stability of the optimal mutant after each round of mutagenesis is as follows: Figure 1 As shown.

[0032] Example 4: Determination of specific enzyme activity of wild-type and mutant trehalose-6-phosphate phosphorylase In a 100 mM HEPES buffer (pH 7.5) containing 10 mM magnesium chloride, 10 mM β-glucose-1-phosphate (βG1P), 10 mM glucose-6-phosphate (G6P), and an appropriate amount of TPP protein solution were added, and the mixture was reacted at 55°C for 10 min. The amount of inorganic phosphate in the reaction solution was measured by a mild pH phosphate assay: 400 μL of the reaction solution was mixed with 4 mL of phosphorus assay reagent (0.1 M zinc acetate, 0.0153 M ammonium molybdate, pH adjusted to 5.0 with hydrochloric acid) and 1 mL of vitamin C solution (100 g / L, pH adjusted to 5.0 with sodium hydroxide), and reacted in a water bath at 30°C for 20 min. The absorbance at 850 nm was measured using a spectrophotometer. Standard concentrations of sodium dihydrogen phosphate solutions (0 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM) were used, and their absorbance at 850 nm was measured according to the steps described above. A standard curve was prepared with absorbance as the ordinate and the concentration of sodium dihydrogen phosphate solution as the abscissa. The concentration of inorganic phosphorus in the enzyme reaction solution was calculated using the standard curve. Under the above measurement conditions, the amount of enzyme required to catalyze the formation of 1 μmol of inorganic phosphate per minute is defined as one unit of enzyme activity (U). Specific enzyme activity is expressed as the enzyme activity per milligram of protein, with units of U / mg.

[0033] Experimental results showed that the specific enzyme activities of wild-type TPP, mutants R1, R2, and R3 were 5.6 U / mg, 5.8 U / mg, 6.7 U / mg, and 9.5 U / mg, respectively. The specific enzyme activity of mutant R3, which exhibited the best thermostability, was 1.7 times that of wild-type TPP.

[0034] Example 5: Discovery and specific activity determination of thermostable maltose phosphorylase and polyphosphate-dependent glucokinase. A search was conducted in the Uniprot database using Maltose phosphorylase as the keyword. Homologous sequences were removed from 979 proteins annotated as Maltose phosphorylase in the search results, and host bacterial growth temperature analysis was performed. Finally, *Thermophilus xiaensis* was identified. Thermophagus xiamenensisThermostable maltose phosphorylase MP (Uniprot ID A0A1I2A9Z4) was obtained from [source missing]. Maltose (final concentration 10 g / L), 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate (pH 7.5), and an appropriate amount of MP protein solution were added to 100 mM HEPES buffer (pH 7.5) containing 10 mM magnesium chloride, and the reaction was carried out at 55°C for 10 min. The specific enzyme activity of MP at 55°C was determined and calculated to be 18.6 U / mg according to literature methods, and the half-life at 60°C was 8.3 h.

[0035] A search was conducted in the Uniprot database using "Polyphosphate glucokinase" as the keyword. Homologous sequences were removed from the 4410 search results, and host bacterial growth temperature analysis was performed to ultimately identify *Nocardia thermophila* (…). Pseudonocardia thermophila The source was polyphosphate-dependent glucokinase PPGK (UniProt ID A0A1M6R178). In a 100 mM HEPES buffer (pH 7.5) containing 10 mM magnesium chloride, glucose (final concentration 2 g / L), 2 mM sodium hexametaphosphate, and an appropriate amount of PPGK protein solution were added. The mixture was reacted at 55°C for 10 min, and samples were taken at different time points to determine the amount of G6P produced. The amount of G6P in the sample was determined as follows: 40 μL of appropriately diluted sample was taken, and 200 μL of G6P assay solution (100 mM HEPES buffer (pH 7.5), 1 mM NAD) was added. + 0.5 U / mL glucose-6-phosphate dehydrogenase was added, and the reaction was carried out at 37℃ for 20 min. The absorbance at 340 nm was measured using a UV spectrophotometer, and the molar extinction coefficient ε was used to determine the absorbance. 340 =6.22 mM - ¹ cm - ¹Calculate the amount of NADH generated. Under the above-described conditions, the amount of enzyme required to catalyze the production of 1 μmol of glucose-6-phosphate per minute is defined as one unit of enzyme activity (U). The amount of glucose-6-phosphate generated is determined by the amount of NADH produced through the glucose-6-phosphate dehydrogenase coupling reaction. Specific enzyme activity is defined as the enzyme activity per unit mass of protein, expressed in U / mg. Experimentally, the specific enzyme activity of PPGK at 55°C is 56.7 U / mg, and its half-life at 60°C is 32.6 h.

[0036] Those skilled in the art can determine the amino acid sequences of the two enzymes based on Unirot numbers A0A1I2A9Z4 and A0A1M6R178, and can also mutate or modify enzymes from these two sources.

[0037] Example 6: Preparation of cells co-expressing three enzymes The MP and PPGK genes were constructed into two multiple cloning sites of the pETDuet plasmid to obtain the pETDuet-mp-ppgk vector, and the gene of the optimal TPP mutant R3 was constructed into the pACYCDuet plasmid to obtain the pACYCDuet-r3 vector. Both vectors were simultaneously transformed into E. coli BL21(DE3) cells, and after induction of expression, the cells were collected to obtain whole cells.

[0038] Example 7 Preparation of whole-cell catalyst Recombinant Escherichia coli BL21(DE3) cells induced to express MP, PPGK, wild-type TPP or mutant R3, as well as the three-enzyme co-expression recombinant cells prepared in Example 6, were collected and suspended in 0.75% sodium chloride solution to OD600=150. Then, each cell suspension was heat-treated in a 60°C water bath for 20 min to obtain the corresponding whole-cell catalysts.

[0039] It should be noted that the proposed solution does not limit the specific heat treatment conditions, as long as the endogenous dephosphorase of Escherichia coli can be inactivated while ensuring that the working enzyme in the whole-cell catalyst maintains a certain working enzyme activity.

[0040] Example 8: Whole-cell catalysis of maltose and sodium hexametaphosphate to synthesize trehalose-6-phosphate A 100 mL reaction mixture containing 200 g / L maltose, 122 g / L sodium hexametaphosphate, 50 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 7.0), 40 mM magnesium sulfate, and whole-cell catalyst was incubated at 55 °C for 18 h. Three sets of experiments were conducted: 1) In Experiment 1, the whole cells contained 10 OD MP, 10 OD PPGK, and 10 OD wild-type TPP; 2) In Experiment 2, the whole cells contained 10 OD MP, 10 OD PPGK, and 10 OD mutant R3; 3) In Experiment 3, the whole cells contained 15 OD three-enzyme co-expression whole cells. All cell addition amounts were calculated based on the final OD600 of the reaction system.

[0041] The above reaction solution was sampled at different time points, and the reaction was terminated by boiling for 5 min. After appropriate dilution, the solution was centrifuged at 12000 rpm for 10 min, and the supernatant was used to determine the concentration of trehalose-6-phosphate by high-performance ion chromatography. The chromatographic column used was an IonPac™ AS11-HC column (250 mm), the mobile phase was 10 mM potassium hydroxide, the flow rate was 1 mL / min, the column temperature was 25℃, and the detector used was a conductivity detector.

[0042] The results are as follows Figure 2As shown, after 2 hours of reaction in Experiment 1, the concentration of T6P was 58.3 g / L, with a molar conversion rate relative to maltose of 23.6%; after 10 hours of reaction in Experiment 2, the concentration of T6P was 232.7 g / L, with a molar conversion rate relative to maltose of 94.3%; and after 15 hours of reaction in Experiment 3, the concentration of T6P was 228.6 g / L, with a molar conversion rate relative to maltose of 92.6%. Scaled up Experiment 3 to a 1-ton reaction vessel, at 80% capacity, after 15 hours of reaction, the concentration of T6P was 216.5 g / L, with a molar conversion rate relative to maltose of 87.7%.

[0043] In summary, this application obtained the optimal mutants R1, R2, and R3 from each round of site-directed and combinatorial mutagenesis. A three-enzyme catalytic system was constructed using wild-type TPP whole cells, MP whole cells, and PPGK whole cells, generating 58.3 g / L T6P at 55℃; while whole cells containing the mutant R3 gene, co-expressing the three enzymes, were able to catalyze the generation of 216.5 g / L T6P in a 1-ton reactor scale-up experiment.

[0044] The catalyst required in this application is whole cell, which can be deactivated by heat treatment of E. coli endogenous dephosphorylase, thereby reducing the dephosphorylation degradation of product T6P; at the same time, heat treatment effectively increases cell permeability and improves the catalytic reaction rate; high-temperature enzyme catalysis at 55°C is beneficial to reduce solution viscosity and improve mass transfer efficiency.

[0045] The heat-resistant trehalose-6-phosphate phosphorylase mutant provided in this application can be used to prepare whole-cell catalysts in one step through heat treatment without the need for enzyme purification, effectively reducing the cost of enzyme use, and can be used as an enzyme preparation for the industrial production of T6P.

[0046] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A thermostable trehalose-6-phosphate phosphorylase mutant, characterized in that, The amino acid sequence of the mutant is obtained by substituting any one of the following amino acids based on the amino acid sequence shown in SEQ ID NO:2: 1) E100M; 2) A106R; 3) T235V; 4) E100M / T235V; 5) E100M / T235V / L580Y; 6) E100M / T235V / T510M; 7) E100M / T235V / T510M / E595A / A702V; 8) E100M / T235V / T510M / E595A / N602P; 9) E100M / T235V / T510M / G430S / A702V; 10) E100M / T235V / T510M / E595P / S607A; 11) E100M / T235V / T510M / G430S / A702V / E595A; 12) E100M / T235V / T510M / G430S / A702V / E595P / S607A; The positions of the amino acid residues are numbered based on the amino acid sequence shown in SEQ ID NO:2, and the slashes indicate that the listed amino acid substitutions are present simultaneously.

2. The thermostable trehalose-6-phosphate phosphorylase mutant according to claim 1, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:

8.

3. A method for preparing the trehalose-6-phosphate phosphorylase mutant as described in claim 1 or 2, characterized in that, A vector containing the gene encoding the trehalose-6-phosphate phosphorylase mutant was introduced into Escherichia coli to obtain recombinant bacteria; the recombinant bacteria were cultured to express the trehalose-6-phosphate phosphorylase mutant.

4. The preparation method according to claim 3, characterized in that, After culturing the recombinant bacteria, the collected recombinant bacteria are subjected to any of the following treatments to obtain a formulation containing the trehalose-6-phosphate phosphorylase mutant: 1) The recombinant bacteria were subjected to heat treatment to obtain a whole-cell catalyst; 2) After cleaving the recombinant bacteria, a crude enzyme solution was obtained. The crude enzyme solution was purified by nickel column chromatography to obtain the trehalose-6-phosphate phosphorylase mutant.

5. A biomaterial, characterized in that, The biomaterial is any one of the following: 1) A nucleic acid molecule, said nucleic acid molecule encoding the trehalose-6-phosphate phosphorylase mutant as described in any one of claims 1 to 2; 2) An expression cassette, the expression cassette comprising the nucleic acid molecule described in item 1) and a promoter operatively linked to the nucleic acid molecule; 3) A recombinant vector comprising the nucleic acid molecule described in item 1) or the expression cassette described in item 2); 4) Recombinant cells, said recombinant cells comprising the nucleic acid molecule described in item 1), the expression cassette described in item 2), or the recombinant vector described in item 3); 5) Whole-cell catalyst, wherein the whole-cell catalyst is prepared by collecting and heat treatment after the recombinant cells described in item 4) express the trehalose-6-phosphate phosphorylase mutant.

6. A method for preparing trehalose-6-phosphate, characterized in that, Using maltose and polyphosphate as substrates, maltose phosphorylase, polyphosphate-dependent glucokinase, and the trehalose-6-phosphate phosphorylase mutant as described in any one of claims 1 to 2 are used to catalyze the reaction to generate trehalose-6-phosphate. The maltose phosphorylase catalyzes the reaction of maltose with inorganic phosphate to produce glucose and β-glucose-1-phosphate, the polyphosphate-dependent glucokinase utilizes polyphosphate as a phosphate donor to catalyze the reaction of glucose with glucose-6-phosphate, and the trehalose-6-phosphate phosphorylase mutant catalyzes the reaction of β-glucose-1-phosphate with glucose-6-phosphate to produce trehalose-6-phosphate.

7. The method for preparing trehalose-6-phosphate according to claim 6, characterized in that, The maltose phosphorylase is *Thermophilus simiensis* (…). Thermophagus xiamenensis (Source) and / or, The polyphosphate-dependent glucokinase is *Pseudomonas thermophila* (…). Pseudonocardia thermophila (Source) 8. The method for preparing trehalose-6-phosphate according to claim 7, characterized in that, The catalytic reaction employs a whole-cell catalyst, which is prepared using *Escherichia coli* as a host and through any of the following methods: 1) Construct three recombinant cells expressing one of the three enzymes respectively, culture and collect each recombinant cell, perform heat treatment separately and then combine them to obtain the whole-cell catalyst; 2) Construct recombinant cells that co-express the three enzymes in the same cell, culture and collect the recombinant cells, and obtain the whole-cell catalyst by heat treatment; 3) Construct a first recombinant cell that co-expresses any two of the three enzymes in the same cell, and a second recombinant cell that expresses the remaining enzyme. Culture and collect the first recombinant cell and the second recombinant cell respectively, perform heat treatment on each cell, and then combine them to obtain the whole-cell catalyst. The three enzymes are maltose phosphorylase, polyphosphate-dependent glucokinase, and trehalose-6-phosphate phosphorylase mutant.

9. The method for preparing trehalose-6-phosphate according to any one of claims 6 to 8, characterized in that, A catalytic reaction is carried out at 50~60℃ to produce trehalose-6-phosphate.

10. The method for preparing trehalose-6-phosphate according to any one of claims 6 to 8, characterized in that, The catalytic system for preparing trehalose-6-phosphate also includes magnesium chloride and / or magnesium sulfate.

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