D-psicose 3-epimerase mutant as well as preparation method and application thereof

Abstract

The invention discloses a D-psicose 3-epimerase mutant as well as a preparation method and application thereof, and relates to the field of gene engineering and enzyme engineering. The amino acid sequence of the D-psicose 3-epimerase mutant is as shown in SEQ ID NO. 1. The invention also discloses a preparation method of the D-psicose 3-epimerase mutant. The specific enzyme activity of the D-psicose 3-epimerase mutant provided by the invention at the temperature of 80 DEG C and the pH value of 6.0 is improved to 15.29 U / mg from 10.21 U / mg of a contrast (before mutation); and when 700g / L fructose is used as a substrate, the equilibrium conversion rate is increased to 36.26% from 29.98% of the contrast (before mutation). The D-psicose 3-epimerase mutant provided by the invention has relatively high catalytic activity, and has a very wide application prospect in efficient production and preparation of D-psicose.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a D-psicose 3-epimerase mutant and a preparation method and application thereof. Background Art

[0002] D-allulose is a low-calorie, rare ketohexose. It boasts 70% of the sweetness of sucrose, yet contains only 10% of its calories, making it an ideal low-energy sweetener alternative. Furthermore, D-allulose possesses a variety of beneficial physiological activities, such as enhancing glucose tolerance, reducing fat accumulation, scavenging reactive oxygen species, and protecting neuronal function. These beneficial effects make it a natural functional ingredient with broad application prospects in food processing and healthcare.

[0003] D-psicose is rare in nature and the cost of direct extraction is high. D-psicose can be obtained through chemical synthesis, but it has many disadvantages such as high substrate price, mixed catalytic products, and environmental pollution. Compared with chemical synthesis, enzymatic biosynthesis of D-psicose has the advantages of high synthesis efficiency, strong substrate specificity, mild reaction conditions, and no environmental pollution. The enzymatic biosynthesis of D-psicose mainly uses D-psicose 3-epimerase to catalyze the conversion of D-fructose to D-psicose. Currently, further improving the catalytic activity of D-psicose 3-epimerase has become the research focus of large-scale synthesis of D-psicose using bioenzymatic methods. Summary of the Invention

[0004] The present invention aims to provide a D-psicose 3-epimerase mutant, its preparation method, and its application to address the aforementioned problems of the prior art. This D-psicose 3-epimerase mutant exhibits high catalytic activity and has broad application prospects in the efficient production of D-psicose.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a D-psicose 3-epimerase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the D-psicose 3-epimerase mutant.

[0008] Furthermore, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector comprising the above-mentioned encoding gene.

[0010] The present invention also provides a recombinant microbial strain comprising the above-mentioned recombinant expression vector.

[0011] The present invention also provides use of the aforementioned encoding gene, recombinant expression vector, or recombinant microbial strain in preparing the D-psicose 3-epimerase mutant according to claim 1.

[0012] The present invention also provides a method for preparing the above-mentioned D-psicose 3-epimerase mutant, comprising the steps of inducing expression and culturing the above-mentioned recombinant microbial strain, and then extracting and purifying to obtain the D-psicose 3-epimerase mutant.

[0013] The present invention also provides use of the D-psicose 3-epimerase mutant in the preparation of D-psicose.

[0014] The present invention also provides a method for preparing D-psicose, comprising the step of utilizing the above-mentioned D-psicose 3-epimerase mutant to catalyze D-fructose to generate D-psicose.

[0015] The present invention also provides a method for improving the catalytic activity of D-psicose 3-epimerase, wherein the amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID NO. 3;

[0016] The method comprises the steps of mutating the 66th amino acid of the D-psicose 3-epimerase into glycine and mutating the 108th amino acid into alanine.

[0017] The present invention discloses the following technical effects:

[0018] The present invention conducts single-site saturation mutagenesis on the amino acid residues involved in binding to the substrate D-fructose in TaDAEase, a D-psicose 3-epimerase derived from a thermophilic archaeon of the family Thermofilaceae. By measuring the enzyme activity of each mutant, single-site mutants C66G and I108A with enhanced enzyme activity were screened. Furthermore, the single-site mutations were integrated to obtain the D-psicose 3-epimerase mutant C66G / I108A with further enhanced enzyme activity.

[0019] The D-psicose 3-epimerase mutant C66G / I108A provided by the present invention has a specific enzyme activity at 80°C and pH 6.0 increased from 10.21 U / mg in the control (before mutation) to 15.29 U / mg. Furthermore, the equilibrium conversion rate using 700 g / L fructose as a substrate increased from 29.98% in the control (before mutation) to 36.26%. The D-psicose 3-epimerase mutant C66G / I108A provided by the present invention has broad application prospects in the efficient production and preparation of D-psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is an SDS-PAGE analysis of D-psicose 3-epimerase TaDAEase and its mutant C66G / I108A;

[0022] Figure 2 HPLC detection chart of standard and D-psicose 3-epimerase TaDAEase enzymatic reaction product;

[0023] Figure 3 HPLC detection chart of the enzymatic reaction products of the standard and D-psicose 3-epimerase mutant C66G / I108A;

[0024] Figure 4 The equilibrium conversion rate of D-psicose synthesized by D-psicose 3-epimerase TaDAEase and mutant C66G / I108A. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The experimental materials involved in the following examples are as follows:

[0031] 1. Strains and vectors

[0032] Escherichia coli JM109, Escherichia coli BL21-CodonPlus (DE3)-RIL and Escherichia coli expression vector pET28a were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0033] 2. Enzymes and other biochemical reagents

[0034] KOD DNA polymerase and KOD-Plus-neo DNA polymerase were purchased from Toyobo, DNA restriction endonucleases and T4 DNA ligase were purchased from Fermentase, DNA gel recovery kit and plasmid extraction kit EZNA were purchased from Omega Bio-tek, and QuickMutation TM Gene site-directed mutagenesis kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., Chelating Sepharose TM Fast Flow was purchased from GE Healthcare, USA. The Bradford protein concentration determination kit was purchased from Shanghai Sangon Biotechnology Co., Ltd. Gene synthesis was performed by Shanghai Boyi Biotechnology Co., Ltd. Polymerase chain reaction primer synthesis and sequencing were performed by Shanghai Sangon Biotechnology Co., Ltd. All other chemical reagents were domestically produced or imported of analytical grade.

[0035] 3. Culture medium

[0036] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0. The screening medium used LB medium containing 50 μg / mL kanamycin.

[0037] The molecular cloning and protein detection techniques used in the present invention are conventional techniques in the art. Techniques not described in detail in the following examples were performed according to the relevant sections of the following laboratory manual: Green MR, Sambrook J. Molecular cloning: a laboratory manual [M]. New York: Cold Spring Harbor Laboratory Press, 2012.

[0038] 4. Amino acid and nucleotide sequences

[0039] The amino acid sequence of the mutant C66G / I108A is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 2. The amino acid sequence of TaDAEase, a D-psicose 3-epimerase from a thermophilic archaeon of the family Thermofilaceae, is shown in SEQ ID NO. 3, and the nucleotide sequence of the gene encoding it is shown in SEQ ID NO. 4. The gene sequence of KpRD, a ribitol dehydrogenase from Klebsiellapneumoniae MGH 48, is shown in SEQ ID NO. 5.

[0040] SEQ ID NO.1:

[0041] MNKIGIYYAYWEHNWAADLLSYPQRVARLGFEILEIKLSVVLAMTERQRRKLKHEAQAHGIGTFGEALDSQIDISSPRPATRKRGIEYLKRGLDTVHKMGGYLLGGALYGAWNLPAVEGMHKAERLMWSVESMRHVLKTAEDVG VICAIEPVNRFEHFMLNTCAEALEYIKMVESPNLGILLDTFHMNIEEDDIYKAIVSAGKNLVHMHVGEPNRKLPGQGRFPWQELLRALRFINYEGAIVMEPFVQVGGEIGLDIKVWRDLARGQDLDEAAQQSLRFLRALLKLSEF.

[0042] SEQ ID NO.2:

[0043] .

[0044] SEQ ID NO.3:

[0045] MNKIGIYYAYWEHNWAADLLSYPQRVARLGFEILEIKLSVVLAMTERQRRKLKHEAQAHGIGLTFCEALDSQIDISSPRPATRKRGIEYLKRGLDTVHKMGGYLLGGILYGAWNLPAVEGMHKAERLMWSVESMRHVLKTAEDVGVICAIEPVNRFEHFMLNTCAEALEYIKMVESPNLGILLDTFHMNIEEDDIYKAIVSAGKNLVHMHVGEPNRKLPGQGRFPWQELLRALRFINYEGAIVMEPFVQVGGEIGLDIKVWRDLARGQDLDEAAQQSLRFLRALLKLSEF.

[0046] SEQ ID NO.4:

[0047] .

[0048] SEQ ID NO.5:

[0049] .

[0050] Example 1 Construction and Screening of Single-Site Mutants of D-psicose 3-epimerase

[0051] 1) Gene synthesis

[0052] Based on the gene sequence of D-psicose 3-epimerase TaDAEase from thermophilic archaea of ​​the Thermofilaceae family, as shown in SEQ ID NO.4, the whole gene of D-psicose 3-epimerase TaDAEase was synthesized by Shanghai Boyi Biotechnology Co., Ltd. to obtain the tadaease gene.

[0053] According to the gene sequence of ribitol dehydrogenase KpRD from Klebsiella pneumoniae MGH 48, as shown in SEQ ID NO.5, the whole gene of ribitol dehydrogenase KpRD was synthesized by Shanghai Boyi Biotechnology Co., Ltd. to obtain the kprd gene.

[0054] 2) Construction of expression vector

[0055] PCR primers F1 and R1 (Table 1) were designed based on the gene sequence of D-psicose 3-epimerase TaDAEase. PCR amplification was performed using the synthetic tadaease gene as a template and primers F1 and R1. PCR amplification conditions were: 98°C for 5 minutes, followed by 30 cycles of 98°C for 20 seconds, 58°C for 40 seconds, and 74°C for 1 minute, and finally 74°C for 10 minutes. The amplified product was double-digested with BamHI and XhoI and ligated into the pET28a vector to construct the recombinant plasmid pET28a-tadaease.

[0056] PCR primers F2 and R2 (Table 1) were designed based on the gene sequence of ribitol dehydrogenase KpRD. PCR amplification was performed using the synthetic kprd gene as a template and primers F2 and R2. PCR amplification conditions were: 98°C for 5 minutes, followed by 30 cycles of 98°C for 20 seconds, 56°C for 40 seconds, and 74°C for 40 seconds, and finally 74°C for 10 minutes. The amplified product was double-digested with Nco I and Not I and ligated into the pET28a vector to construct the recombinant plasmid pET28a-kprd.

[0057] Table 1 Primers used to construct recombinant plasmids

[0058]

[0059] Note: The underlined part is the restriction enzyme cleavage site.

[0060] 3) Construction of D-psicose 3-epimerase TaDAEase mutant

[0061] Selection of Mutation Sites: Using the tertiary structure of D-psicose 3-epimerase from Agrobacterium tumefaciens (PDB ID: 2HK1) as a template, the three-dimensional molecular structure of TaDAEase (D-psicose 3-epimerase) was modeled using Swiss-Model (http: / / swissmodel.expasy.org). The TaDAEase protein structure was obtained by docking with the substrate D-fructose using AutoDock 4.2 software. The amino acid residues involved in binding to the substrate D-fructose were identified. Non-conserved amino acid residues Y7, C66, and I108 were selected for site-directed saturation mutagenesis to construct saturation mutants.

[0062] Construction of site-directed saturation mutants: according to QuickMutation TM The instructions of the gene site-directed mutagenesis kit were used to design primers based on the base sequence of the gene tadaease and the amino acid site to be mutated, as shown in Table 2. The specific steps for constructing the saturation mutant are as follows: (1) Taking the construction of mutant Y7G as an example, the recombinant plasmid pET28a-tadaease was used as a template, and primers Y7G-F and Y7G-R were used according to the QuickMutation TM PCR amplification was performed according to the instructions of the gene site-directed mutagenesis kit. The amplified product was treated with Dpn I enzyme and transformed into Escherichia coli JM109. Transformants were selected on kanamycin-resistant plates, and the recombinant plasmid was extracted. The recombinant plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing and alignment with the corresponding gene sequence, confirming the successful construction of the recombinant plasmid pET28a-tadaeaseY7G. The construction of the site-directed mutants shown in Table 2 was based on the construction method of mutant Y7G.

[0063] Table 2 Primers used to construct site-directed mutants

[0064]

[0065]

[0066]

[0067]

[0068] Note: The part marked with a box is the mutated base.

[0069] 4) Screening of D-psicose 3-epimerase TaDAEase mutants

[0070] Mutant Expression: Mutant expression plasmids were transformed into Escherichia coli BL21-CodonPlus(DE3)-RIL. Single mutant clones were selected from fresh transformation plates and transferred to each well of a 48-well microplate containing 300 μL of LB medium (50 μg / mL kanamycin). The microplate was shaken at 37°C and 200 rpm for 12 hours to obtain seed culture. The seed culture was transferred to a new 48-well microplate containing 300 μL of LB medium (50 μg / mL kanamycin) at a 1% inoculum level. The culture was shaken at 37°C and 200 rpm for 3 hours. IPTG was added to each well to a final concentration of 0.25 mmol / L. The culture was continued at 16°C and 200 rpm for 18 hours. After the incubation period, the microplate was centrifuged at 4000 rpm for 15 minutes to collect the cells. 300 μL of 50 mmol / L MES buffer (pH 6.0) was added to each well to resuspend the cells. Then, 20 μL of 20 mg / mL lysozyme was added to each well. The microplate was incubated at 37°C and 200 rpm for 2 hours to lyse the cells and release the recombinant enzyme. After the reaction was complete, the microplate was centrifuged at 4000 rpm for 15 minutes. The supernatant obtained after centrifugation was the crude enzyme solution of the mutant.

[0071] Expression of ribitol dehydrogenase KpRD: The expression vector pET28a-kprd of ribitol dehydrogenase KpRD was transformed into Escherichia coli BL21-CodonPlus (DE3) -RIL and spread on LB solid medium containing 50μg / mL kanamycin. A single colony was picked from the transformation plate and inoculated into 10mL of LB liquid medium containing 50μg / mL kanamycin, and cultured at 37℃ and 200r / min for 12h. The activated culture was transferred to 100mL of LB liquid medium containing 50μg / mL kanamycin at a 1% inoculum volume, and cultured at 37℃ and 200r / min until the OD value of the culture solution reached 0. 600nm The concentration of IPTG was 0.6, and IPTG was added thereto to a final concentration of 0.5mmol / L, and then the shaking culture was continued at 30℃ and 200r / min for 6h. After the expression was completed, the bacterial solution was centrifuged at 4℃ and 4000r / min for 10min, the bacterial precipitate was collected, and the bacterial precipitate was washed with 50mmol / L MES buffer (pH 6.0). 50mmol / LMES buffer (pH 6.0) was added to the bacteria collected by centrifugation, the bacteria were resuspended, and placed on ice to crush the cells with ultrasound. The parameters of the ultrasonic cell crusher were set as follows: ultrasonic power was 25%, ultrasonic crushing time was 3sec, and interval was 6sec. The bacterial cells were treated with ultrasound until the bacterial suspension became a uniform solution. The cell rupture solution was centrifuged at 4℃ and 8000r / min for 10min, the supernatant was collected, and impurities were removed by passing through a 0.45μm water filter membrane to obtain a crude enzyme solution. Using Ni2+ The target protein was purified from the crude enzyme solution using an affinity chromatography column and eluted with 200 mM imidazole elution buffer to obtain a purified KpRD enzyme solution. The purified KpRD enzyme solution was dialyzed against 50 mmol / L MES buffer (pH 6.0) and diluted to 0.5 mg / mL using 50 mmol / L MES buffer (pH 6.0).

[0072] Screening of mutants: Take a 96-well microplate, add 20μL of mutant crude enzyme solution and 100μL of D-fructose with a mass concentration of 24g / L to each well, and add CoCl2 solution to a final concentration of 1mmol / L, and react the microplate at 80℃ for 30min. Immediately add 120μL of KpRD enzyme solution with a mass concentration of 0.5mg / mL to each well and add NADH to a final concentration of 5mmol / L. Place the microplate at 30℃ and monitor the change in absorbance of the reaction system at 340nm at 0min and 30min over time. The change in absorbance of the reaction system at 340nm at 0min and 30min over time is proportional to the yield of D-psicose in the reaction system. The △A of the reaction system at 0min and 30min 340nm The larger the value, the higher the yield of D-psicose and the higher the enzyme activity of the mutant.

[0073] The reaction system corresponding to the D-psicose 3-epimerase mutant was at △A at 0 min and 30 min. 340nm The results of the value determination are shown in Table 3. As shown in Table 3, the enzymatic activities of the site-directed mutants related to tyrosine 7 (Y7) in D-psicose 3-epimerase TaDAEase were all lower than those of TaDAEase; the enzymatic activity of the site-directed mutant C66G related to cysteine ​​66 (C66) in TaDAEase was higher than that of TaDAEase; the enzymatic activities of the site-directed mutants I108A, I108P, and I108F related to isoleucine 108 (I108) in TaDAEase were higher than those of TaDAEase, and the enzymatic activity of mutant I108A was the highest.

[0074] Table 3 ΔA of the reaction system at 0 min and 30 min 340nm Value measurement results

[0075]

[0076]

[0077] Example 2 Construction and enzyme activity determination of mutant C66G / I108A

[0078] 1) Construction of mutant C66G / I108A

[0079] Using the recombinant plasmid pET28a-tadaeaseC66G as a template and primers I108A-F and I108A-R, the QuickMutation TM PCR amplification was performed according to the instructions of the gene site-directed mutagenesis kit. The amplified product was treated with Dpn I enzyme and transformed into Escherichia coli JM109. Transformants were selected on kanamycin-resistant plates, and the recombinant plasmid was extracted. The recombinant plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing and comparison with the corresponding gene sequence confirmed the successful construction of the recombinant plasmid pET28a-tadaeaseC66G / I108A.

[0080] 2) Expression and purification of D-psicose 3-epimerase TaDAEase and its mutant C66G / I108A

[0081] The recombinant plasmids pET28a-tadaease and pET28a-tadaeaseC66G / I108A were transformed into Escherichia coli BL21-CodonPlus (DE3) -RIL, respectively, and spread on LB solid medium containing 50 μg / mL kanamycin. Single colonies were picked from the transformation plate and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 200 r / min for 12 hours. The activated cultured bacterial solution was transferred to 100 mL of LB liquid medium containing 50 μg / mL kanamycin at a 1% inoculum volume, and cultured at 37°C and 200 r / min until the bacterial solution OD 600nm The pH value was 0.4, IPTG was added thereto to a final concentration of 0.25 mM, and then shaking culture was continued at 16°C and 200 r / min for 12 h.

[0082] After the expression is completed, the bacterial solution is centrifuged at 4°C and 4000r / min for 10 minutes, the bacterial precipitate is collected, and the bacterial precipitate is washed with 50mmol / LMES buffer (pH 6.0). 50mmol / L MES buffer (pH6.0) is added to the bacteria collected by centrifugation, the bacteria are resuspended, and placed on ice to disrupt the cells with ultrasound. The parameters of the ultrasonic cell disruptor are set as follows: ultrasonic power is 25%, ultrasonic disruption time is 3sec, and interval is 6sec. Ultrasonic treatment of bacterial cells until the bacterial suspension becomes a uniform solution. The cell disruption solution is centrifuged at 4°C and 8000r / min for 10 minutes, the supernatant is collected, and impurities are removed by passing through a 0.45μm water filter membrane to obtain a crude enzyme solution. Using Ni 2+The target protein in the crude enzyme solution was purified using an affinity chromatography column and eluted with 250 mM imidazole elution buffer to obtain a purified enzyme solution. The purified enzyme solution was dialyzed against 50 mmol / L MES buffer (pH 6.0). The enzyme purity was determined by SDS-PAGE. The SDS-PAGE analysis of D-psicose 3-epimerase TaDAEase and mutant C66G / I108A is shown in Figure 2. Figure 1 shown.

[0083] 3) Determination of D-psicose 3-epimerase TaDAEase and its mutant C66G / I108A activity

[0084] 10 μL of the purified enzyme solution was mixed with 990 μL of 50 mmol / L MES buffer (pH 6.0) containing 50 g / L D-fructose and 1 mmol / L CoCl2. The mixture was reacted at 80°C for 30 minutes, then quickly placed in a boiling water bath for 5 minutes to terminate the reaction. The reaction solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove impurities. A high-performance liquid chromatography (HPLC) sample was prepared, and the concentration of the product, D-psicose, was determined by HPLC.

[0085] High-performance liquid chromatography (HPLC) was performed using a Carbohydrate ES-w column (5 μm, 4.6 × 250 mm) and a 1260 Infinity Evaporative Light Scattering Detector. HPLC conditions were: 75% acetonitrile and 25% deionized water as the mobile phase, a flow rate of 1.0 mL / min, a column temperature of 40°C, a carrier gas pressure of 30 psi, a drift tube temperature of 55°C, an injection volume of 10 μL, and a run time of 22 min per sample.

[0086] Definition of enzyme activity unit (U): The amount of enzyme required to convert D-fructose to 1 μM D-psicose per minute is one enzyme activity unit (U).

[0087] The enzyme activity results of the D-psicose 3-epimerase TaDAEase and mutant C66G / I108A are shown in Table 4. The enzyme activity of TaDAEase was 10.21 U / mg, and that of mutant C66G / I108A was 15.29 U / mg. Compared to TaDAEase, the activity of mutant C66G / I108A was 0.50-fold higher.

[0088] Table 4 Enzyme activity assay results of TaDAEase and mutant C66G / I108A

[0089]

[0090] Example 3 Application of D-psicose 3-epimerase TaDAEase and C66G / I108A in the preparation of D-psicose

[0091] A 1 mL reaction system was prepared containing an enzyme solution (the purified D-psicose 3-epimerase TaDAEase and the mutant C66G / I108A enzyme solution prepared in Example 2), a substrate D-fructose, and CoCl2. The enzyme solution was added in an amount of 10 U / mL, the final concentration of D-fructose was 100 g / L, the final concentration of CoCl2 was 1 mmol / L, and the buffer system was 50 mmol / L MES buffer (pH 6.0). After the reaction solution was reacted at 80° C. for 5 h, it was quickly placed in a boiling water bath for 5 min to terminate the enzyme reaction. The reaction solution was then centrifuged at 12,000 r / min for 10 min, the supernatant was taken, and filtered through a 0.22 μm water filter membrane to remove impurities to prepare a high-performance liquid chromatography test sample. The concentration of the product D-psicose was determined by high-performance liquid chromatography using the same method as in Example 2.

[0092] HPLC detection of standard and D-psicose 3-epimerase TaDAEase enzymatic reaction products as shown in the figure Figure 2 As shown; HPLC detection of standard and mutant C66G / I108A enzymatic reaction products as shown Figure 3 As shown in the figure, the D-psicose concentration in the reaction product catalyzed by TaDAEase was 30.09 mg / mL; the D-psicose concentration in the reaction product catalyzed by mutant C66G / I108A was 39.63 mg / mL. Mutant C66G / I108A catalyzed the conversion of D-fructose to D-psicose at a higher yield.

[0093] Example 4 Equilibrium Conversion Rate of D-Psicose 3-Epimerase TaDAEase and Mutant C66G / I108A to High Concentration D-Fructose

[0094] A 50 mL reaction system was prepared containing an enzyme solution (the purified D-psicose 3-epimerase TaDAEase and the mutant C66G / I108A enzyme solution prepared in Example 2), the substrate D-fructose, and CoCl2. The enzyme solution was added at 10 U / mL, the final concentration of D-fructose was 700 g / L, the final concentration of CoCl2 was 1 mmol / L, and the buffer system was 50 mmol / L MES buffer (pH 6.0). The reaction solution was reacted at 80°C, and samples were taken at regular intervals. The concentration of the product D-psicose was determined by high-performance liquid chromatography (using the same method as in Example 2), and the equilibrium conversion rate of D-psicose was calculated.

[0095] The equilibrium conversion rate was calculated by dividing the D-psicose concentration in samples at different time points by the concentration of the substrate D-fructose added before the start of the reaction: equilibrium conversion rate (%) = (D-psicose concentration) / (substrate D-fructose concentration) × 100%.

[0096] The equilibrium conversion rate of D-psicose 3-epimerase TaDAEase and mutant C66G / I108A to 700 g / L D-fructose was determined as follows: Figure 4 The equilibrium conversion rate of D-psicose 3-epimerase TaDAEase on 700 g / L D-fructose was 29.98%, while the equilibrium conversion rate of mutant C66G / I108A on 700 g / L D-fructose was 36.26%, an increase of 20.94% compared to TaDAEase.

[0097] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A D-psicose 3-epimerase mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A gene encoding the D-psicose 3-epimerase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: Comprising the coding gene according to claim 2 or 3.

5. A recombinant microbial strain, characterized in that Comprising the recombinant expression vector according to claim 4.

6. Use of the encoding gene according to claim 2 or 3, the recombinant expression vector according to claim 4, or the recombinant microbial strain according to claim 5 in preparing the D-psicose 3-epimerase mutant according to claim 1.

7. A method for preparing the D-psicose 3-epimerase mutant according to claim 1, characterized in that: The method comprises the steps of inducing expression culture of the recombinant microbial strain according to claim 5, and then extracting and purifying the D-psicose 3-epimerase mutant.

8. Use of the D-psicose 3-epimerase mutant according to claim 1 in the preparation of D-psicose.

9. A method for preparing D-psicose, characterized in that: The method comprises the step of using the D-psicose 3-epimerase mutant according to claim 1 to catalyze the production of D-psicose from D-fructose.

10. A method for improving the catalytic activity of D-psicose 3-epimerase, characterized in that: The amino acid sequence of the D-psicose 3-epimerase is shown in SEQ ID NO. 3; The method comprises the steps of mutating the 66th amino acid of the D-psicose 3-epimerase into glycine and mutating the 108th amino acid into alanine.

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