Ribose-5-phosphate isomerase mutant and application thereof in large-scale production of allose

Abstract

The invention discloses a ribose-5-phosphate isomerase mutant and application of the ribose-5-phosphate isomerase mutant in large-scale production of allose, and belongs to the technical field of molecular biology. According to the high-activity ribose-5-phosphate isomerase mutant disclosed by the invention, Q56R / K79E / G130W triple mutation is carried out on ribose-5-phosphate isomerase, then pET-28a (+) is taken as an expression vector, escherichia coli BL21 is adopted for expression, and the final enzyme activity reaches 1048U / mL, which is improved by two times compared with the original enzyme protein activity. When the enzyme is applied to immobilized catalysis of allose production, the allose conversion rate is 36%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, and more particularly to a ribose-5-phosphate isomerase mutant and its application in large-scale production of allose. BACKGROUND

[0002] Ribose-5-phosphate isomerase (RpiB, EC 5.3.1.6) is a key enzyme that catalyzes the reversible isomerization of D-ribose-5-phosphate and D-ribulose-5-phosphate. In recent years, it has attracted much attention due to its potential application value in the biosynthesis of allose. However, there is still a significant gap between the catalytic performance of RpiB in the prior art and the industrialization demand.

[0003] The specific activity of wild-type RpiB (derived from WP_012002392.1 of Pseudo-thermomyces) is generally lower than 500 U / mg. The specific activity of the native enzyme under the condition of 50℃ and pH 7.0 is only 418±22 U / mg, and the conversion rate of D-allose is less than 25%. Moreover, the wild-type enzyme rapidly inactivates at a temperature above 50℃, with a half-life (t1 / 2) of less than 3 hours. Frequent enzyme supplementation is required under high-temperature reaction, which increases the production cost.

[0004] In industrial production, the existing enzymatic process has an allose conversion rate of only 28-30% at a substrate concentration of 30%, and requires the addition of a high concentration of co-factor (such as 2mM Co 2+ ), which increases the difficulty of purification. The substrate inhibition effect is significant, and when the substrate concentration is >20%, the reaction rate decreases by 40%. In traditional batch reactions, free enzymes cannot be reused, resulting in enzyme costs accounting for more than 35% of the total cost.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a ribose-5-phosphate isomerase mutant and its application in large-scale production of allose. SUMMARY

[0006] Therefore, the present application provides a ribose-5-phosphate isomerase mutant and its application in large-scale production of allose, which solves the problems of low enzyme activity of ribose-5-phosphate isomerase and low conversion rate of allose production catalyzed by the enzyme using allosone as a substrate.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0008] A mutant sequence for synthesizing ribose-5-phosphate isomerase in Escherichia coli, the protein sequence of the ribose-5-phosphate isomerase mutant is shown in SEQ ID NO. 1. Based on the amino acid sequence of SEQ ID NO. 3, it contains Q56R, K79E and G130W mutations.

[0009] The nucleotide sequence of the ribose-5-phosphate isomerase mutant is shown as SEQ ID NO. 2.

[0010] A sequence for synthesizing ribose-5-phosphate isomerase in Escherichia coli, and the protein sequence of the ribose-5-phosphate isomerase is shown as SEQ ID NO. 3.

[0011] The nucleotide sequence of the ribose-5-phosphate isomerase is shown as SEQ ID NO. 4.

[0012] A recombinant vector of a ribose-5-phosphate isomerase mutant gene and a recombinant Escherichia coli strain containing the recombinant vector.

[0013] A recombinant Escherichia coli strain, which expresses ribose-5-phosphate isomerase derived from Pseudothermotoga, and the nucleotide sequence of the ribose-5-phosphate isomerase is shown as SEQ ID NO. 2 or SEQ ID NO. 4.

[0014] The recombinant Escherichia coli takes BL21 as the expression host and takes pET-28a(+) plasmid as the expression vector.

[0015] The ribose-5-phosphate isomerase mutant, the expression vector or the recombinant bacteria are applied in the large-scale production of allose.

[0016] The ribose-5-phosphate isomerase mutant, the expression vector or the recombinant bacteria are applied in improving the enzyme activity of ribose-5-phosphate isomerase.

[0017] An immobilized enzyme microsphere is prepared by cross-linking the ribose-5-phosphate isomerase mutant with calcium alginate and chitosan.

[0018] The ribose-5-phosphate isomerase mutant fermentation broth is mixed with 2% (w / v) sodium alginate + 0.1% (w / v) chitosan, cross-linked with CaCl2 into 3mm microspheres, and subjected to allose production with allose as the substrate, and the conversion rate is 36%.

[0019] The immobilized enzyme microsphere is applied in the large-scale production of allose.

[0020] Compared with the prior art, the ribose-5-phosphate isomerase mutant and its application in large-scale production of allose are provided, the RpiB enzyme is rationally designed to enhance the substrate binding capacity (reduce Km) and thermal stability (extend t1 / 2) through enzyme molecular modification, the genetically engineered bacteria BL21 / pET-28a(+)-RpibMUT is successfully constructed, the soluble expressed recombinant enzyme is obtained, the recombinant enzyme has the characteristics of good heat resistance, no need of metal ion assistance, no generation of D-arabinose by-product, etc. The low-cost immobilized carrier is developed, the enzyme activity recovery rate is > 90% and the reuse is ≥ 8 times, the substrate inhibition effect is broken through, the conversion rate is maintained ≥ 35% under 30% high concentration, and the recombinant enzyme has good industrial application potential. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0022] Figure 1 The plasmid map of the recombinant plasmid pET-28a(+)-RpiB and pET-28a(+)-RpiBMUT;

[0023] Figure 2 The SDS-PAGE map of the recombinant pET-28a(+)-Rpib enzyme and pET-28a(+)-RpibMUT enzyme, the first lane is a protein molecular weight marker, the second lane is the recombinant RpibMUT enzyme, and the third lane is the recombinant Rpib enzyme;

[0024] Figure 3 The bioconversion result of D-allose; the retention time is 4.8 min: the phosphate peak; 13.37 min: the allose peak; 17.3 min: the psicose peak. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0026] The ribose-5-phosphate isomerase nucleotide sequence (as shown in SEQ ID NO. 4) and the ribose-5-phosphate isomerase mutant nucleotide sequence (as shown in SEQ ID NO. 2) are fully synthesized by Beijing Qikexin Biotechnology Co., Ltd.

[0027] The ribose-5-phosphate isomerase mutant amino acid sequence is shown in SEQ ID NO. 1.

[0028] MKIAIGCDHAGFKLKEAIKGYLVSKGFNILDEGTYSEDSVDYPDFAAK VALDIKN R RADFGILICGTGIGMSIAANRI E GIRAALCLFPEMAKLARSHNN ANILVLPGRFIAVELAQWIVDAFIEEKFE W GRHENRVQKIEEMDK; SEQ ID NO. 1.

[0029] The ribose-5-phosphate isomerase mutant nucleotide sequence is shown in SEQ ID NO. 2.

[0030] ATGAAAATAGCAATTGGATGTG ATCACGCTGGCTTTAAGCTGAAAGAAGCAATTAAGGGTTATCTGGTCAGCAAAGGCTTCAACATCCTTGACGAAGGCACCTACTCTGAAGATAGCGTTGACTACCCGGATTTTGCTGCGAAGGTGGCACTGGACATCAAGAAC CGC CGTGCGGATTTCGGCATCCTGATTTGCGGTACGGGTATTGGTATGAGCATTGCTGCCAATCGTATT GAG GGCATCCGCGCGGCCCTGTGTTTGTTTCCGGAAATGGCAAAGCTCGCGCGTTCCCATAATAACGCGAACATCTTGGTGCTGCCGGGTCGCTTCATCGCTGTGGAACTGGCGCAGTGGATTGTTGACGCGTTTATCGAGGAGAAATTCGAG TGG GGTCGTCACGAAAACCGTGTT CAAAAAATCGAGGAGATGGATAAA ; SEQ ID NO. 2.

[0031] The ribose-5-phosphate isomerase amino acid sequence is shown in SEQ ID NO. 3.

[0032] MKIAIGCDHAGFKLKEAIKGYLVSKGFNILDEGTYSEDSVDYPDFAAK VALDIKN Q RADFGILICGTGIGMSIAANRI K GIRAALCLFPEMAKLARSHNN ANILVLPGRFIAVELAQWIVDAFIEEKFE G GRHENRVQKIEEMDK;SEQ ID NO.3.

[0033] The nucleotide sequence of ribose-5-phosphate isomerase is shown in SEQ ID NO. 4.

[0034] ATGAAAATAGCAATTGGATGTG ATCACGCTGGCTTTAAGCTGAAAGAAGCAATTAAGGGTTATCTGGTCAGCAAAGGCTTCAACATCCTTGACGAAGGCACCTACTCTGAAGATAGCGTTGACTACCCGGATTTTGCTGCGAAGGTGGCACTGGACATCAAGAAC CAG CGTGCGGATTTCGGCATCCTGATTTGCGGTACGGGTATTGGTATGAGCATTGCTGCCAATCGTATT AAG GGCATCCGCGCGGCCCTGTGTTTGTTTCCGGAAATGGCAAAGCTCGCGCGTTCCCATAATAACGCGAACATCTTGGTGCTGCCGGGTCGCTTCATCGCTGTGGAACTGGCGCAGTGGATTGTTGACGCGTTTATCGAGGAGAAATTCGAG GGC GGTCGTCACGAAAACCGTGTT CAAAAAATCGAGGAGATGGATAAA ; SEQ ID NO. 4.

[0035] The present application uses the conventional techniques and methods used in the field of genetic engineering and molecular biology, and uses Gibson ligase to connect ribose-5-phosphate isomerase and ribose-5-phosphate isomerase mutant at 50°C for 30 minutes, then transform E. coli BL21 competent cells to obtain recombinant bacteria BL21-pET-28a(+)-Rpib and BL21-pET-28a(+)-RpibMUT.

[0036] The base plasmid pET-28a(+) and BL21 E. coli were purchased from Mingling Biotechnology Co., Ltd.; other reagents involved in the examples were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0037] LB liquid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0038] LB solid medium: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L, agar powder 20 g / L.

[0039] M9 medium (fermentation base medium): glucose 5.0 g / L, MgSO4·7H2O 0.5 g / L, Na2HPO4·12H2O 17.1 g / L, ammonium chloride 1.0 g / L, sodium chloride 0.5 g / L, anhydrous KH2PO4 3.0 g / L.

[0040] D-allose determination method: D-psicose and D-allose standard stock solutions with a concentration of 10 g / L were prepared, respectively, and then diluted with double deionized water to 2, 4, 6, 8 g / L, respectively. Finally, the prepared standard solutions with different concentrations were filtered into liquid phase vials through a 0.22 mm water filter, and detected and analyzed by HPLC. The HPLC detection conditions are shown in Table 1.

[0041] Table 1

[0042]

[0043] HPLC analysis was performed to obtain the peak area corresponding to the standard solution with different concentrations. Therefore, the concentration was taken as the abscissa, and the peak area corresponding to each concentration standard solution was taken as the ordinate to draw the standard curve. The D-psicose standard curve was Y = 166647X - 1001.67 (R 2 = 0.99997), and the D-allose standard curve was Y = 162414X - 89126.3 (R 2 = 0.99995). When the peak area of the detected substance is known, the corresponding concentration can be obtained according to the standard curve.

[0044] Immobilization method: the fermentation broth was mixed with 2% (w / v) sodium alginate + 0.1% (w / v) chitosan solution, and cross-linked into 3 mm microspheres by CaCl2.

[0045] Example 1 Construction of recombinant expression vectors pET-28a(+)-Rpib and pET-28a(+)-RpibMUT and obtaining of recombinant bacteria BL21-pET-28a(+)-Rpib and BL21-pET-28a(+)-RpibMUT

[0046] The Rpib and RpibMUT sequences were respectively connected to the pET-28a(+) vector by homologous recombination to obtain the recombinant vectors pET-28a(+)-Rpib and pET-28a(+)-RpibMUT. The primers are shown in Table 2.

[0047] Table 2 primer sequences

[0048]

[0049]

[0050] The Rpib and RpibMUT fragments were respectively amplified using the primers gene-F / R with the full gene synthesized Rpib sequence (as shown in SEQ ID NO. 4) and RpibMUT sequence (as shown in SEQ ID NO. 2) as templates; the vector sequence was amplified using the primers vector-F / R with the pET-28a(+) vector as template. The PCR reaction system is shown in Table 3. The PCR program was 98℃ 2min; 98℃ 30s, 60℃ 30s, 72℃ 30s, 30 cycles; 72℃ 10min.

[0051] Table 3 PCR reaction system

[0052]

[0053] The homologous recombination was performed according to the following method (Wuhan Junno De Biotechnology Co., Ltd., one-step homologous recombination directional cloning kit, item number V6006): the homologous recombination reaction system is shown in Table 4. The PCR program was 50℃ 30min.

[0054] Table 4 homologous recombination reaction system

[0055]

[0056] The transformation system was added to BL21 competent cells, ice bath for 30min, 42℃ heat shock for 90s, added with 5ml LB liquid medium, 37℃ 150r / min culture for 90min, completely spread on LB solid plate containing 100mg / ml kanamycin, after 16h, single colony was picked and expanded to obtain the recombinant expression vectors pET-28a(+)-Rpib and pET-28a(+)-RpibMUT (vector map is shown in Figure 1 ) and the recombinant bacteria BL21-pET-28a(+)-Rpib and BL21-pET-28a(+)-RpibMUT.

[0057] Example 2 Fermentation of recombinant bacteria BL21-pET-28a(+)-Rpib and BL21-pET-28a(+)-RpibMUT

[0058] Select a single transformant and propagate it into a 1L LB shake flask seed culture. OD 600 =0.8, all transferred to a 50L fermenter (containing the basal fermentation medium), OD 600 At 8 o'clock, IPTG was added to a final concentration of 0.1 mmol / L for induction. Fermentation was carried out at 27°C and 300 rpm for 24 hours to obtain the fermentation broth. After SDS-PAGE protein electrophoresis... Figure 2 Enzyme activity assay:

[0059] Crude enzyme extraction method: Centrifuge the fermentation broth (4500 r / min, 5 min), wash twice with 50 mmol / L pH 7.4 PBS buffer, remove the supernatant to obtain bacterial cells; then add PBS buffer at a ratio of 1g bacterial cells: 20mL PBS buffer to resuspend the bacterial cells, and sonicate to disrupt the broth (power 350W, sonication for 5s, pause for 10s, working time 30min), centrifuge and retain the supernatant to obtain crude enzyme solution.

[0060] Reaction system: 50mM sodium phosphate buffer (pH 7.2), 2mM MnCl2, 5mM ribose-5-phosphate substrate, 1% crude enzyme solution added, reacted at 55℃ for 10 min.

[0061] DNS method for enzyme activity determination: Enzyme activity is defined as the amount of enzyme required to produce 1 μmol allose per minute. The results are shown in Table 5.

[0062] Table 5 Comparison of Enzyme Properties

[0063]

[0064] Example 3: Scaled-up reaction and product analysis

[0065] Sodium alginate (2% w / v) and chitosan (0.1% w / v) were dissolved in 50 mM phosphate buffer (pH 7.0), and crude enzyme solution (enzyme concentration 50 mg / mL) was added. The mixture was magnetically stirred for 2 h to form a homogeneous suspension. The mixture was then dropped dropwise into a 1% CaCl2 solution crosslinking bath using a microsphere generator (nozzle diameter 0.5 mm) at a dropping rate of 10 mL / min and a drop height of 15 cm. After standing in the crosslinking bath for 30 min, the microspheres were washed three times with deionized water and then freeze-dried under vacuum (-50℃, 24 h) to obtain dried immobilized enzyme microspheres. A packed bed reactor (diameter 20 cm, height 50 cm) was used, and 5 kg of immobilized enzyme microspheres (bulk density 0.65 g / cm³) were packed into the reactor. 3), the substrate solution (30% D- Psicose) was circulated countercurrently at a flow rate of 0.8 BV / h (Bed Volume / hour). After 6h, the reaction solution contained 36% D-Aloketose and 63% D- Psicose, i.e. the enzyme catalyzed the keto-enol isomerization between D-Aloketose and D-Aloketose reached an equilibrium of 36:63, see Figure 3 .

[0066] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Numerous modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Therefore, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A ribose-5-phosphate isomerase mutant, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The ribose-5-phosphate isomerase mutant according to claim 1, characterized in that, The nucleotide sequence of its encoding gene is shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, It comprises the nucleotide sequence of claim 2.

4. A recombinant bacterium, characterized in that, It includes the expression vector as described in claim 3.

5. The application of the ribose-5-phosphate isomerase mutant according to any one of claims 1-2, the expression vector according to claim 3, or the recombinant bacteria according to claim 4 in the large-scale production of allosugar.

6. The use of the ribose-5-phosphate isomerase mutant according to any one of claims 1-2, the expression vector according to claim 3, or the recombinant bacteria according to claim 4 in improving the activity of ribose-5-phosphate isomerase.

7. An immobilized enzyme microsphere, characterized in that, It is prepared by cross-linking the ribose-5-phosphate isomerase mutant of claim 1 with calcium alginate and chitosan.

8. The application of the immobilized enzyme microspheres according to claim 7 in the large-scale production of allosugar.

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