A L-rhamnose sugar kinase mutant and preparation method thereof

Abstract

The present invention discloses an L-rhamnose gum sugar kinase mutant and a preparation method thereof, and belongs to the field of enzyme engineering. The present invention obtains 4 mutants with improved enzyme activity or thermal stability; among them, the enzyme activity of mutant N65L is 1.15 times that of the wild type compared with the wild type. In addition, after the mutants A59W and Q147H were incubated at 50°C for a certain period of time, the residual enzyme activities were 1.54 and 1.33 times of the wild type, respectively; after E24G, A59W and Q147H were incubated at 70°C for a certain period of time, the residual enzyme activities were 2.25, 1.83 and 1.60 times of the wild type, respectively. The present invention obtains an L-rhamnose gum sugar kinase mutant with improved enzyme activity and thermal stability, which provides an effective way to improve the efficiency of catalyzing the synthesis of rare sugars, and has great application potential and economic value in industrial production.

Description

Technical Field

[0001] The invention belongs to the field of enzyme engineering, and particularly relates to an L-rhamnose glycokinase mutant and a preparation method thereof. Background Art

[0002] L-rhamnulose kinase (RhaB) is a member of the hexokinase-hsp70-superprotein family. It has five β-folds at the N-terminus and C-terminus and contains ATP binding sites. It is a metal-dependent enzyme. As a substrate-specific kinase, RhaB is also one of the key enzymes required for the metabolism of L-rhamnose in Escherichia coli. It can catalyze substrates with rare sugars with R-type C-3 configuration, such as D-psicose, and can phosphorylate D-psicose to produce D-psicose-1-phosphate, while consuming one molecule of ATP to generate the corresponding ADP. This reaction is an irreversible process and has no catalytic activity for sugars with S-type C-3 configuration.

[0003] Therefore, in recent years, some studies have applied RhaB to the synthesis of rare sugars, such as in the synthesis of psicose. At present, the main method for the synthesis of D-psicose is to use D-psicose 3-epimerase (DAE) to catalyze the direct conversion of D-fructose into D-psicose, but this reaction is a reversible process with a low conversion rate, which seriously limits its industrial application. By connecting isomerase and RhaB in series, the phosphorylation reaction of the latter is used to break the bottleneck of the isomerization reaction equilibrium, thereby increasing the yield of D-psicose and facilitating industrial production. However, there are still few types of RhaB that have been explored and applied, and the performance of the enzyme needs to be improved. Therefore, in order to better realize the industrial production of rare sugars, screening out RhaB mutants with strong activity and good thermal stability plays an important role in improving its industrial application efficiency and promoting the development of related industries. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide an L-rhamnose kinase mutant with improved enzyme activity or thermal stability and a preparation method thereof, so as to solve the problem that the activity of the existing rare sugar synthesis phosphorylation pathway is not ideal and it is difficult to achieve industrial production.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The present invention provides a L-rhamnose kinase mutant with improved enzyme activity or thermal stability, wherein the amino acid sequence of the mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations:

[0007] At least one of E24G, A59W, N65L and Q147H; wherein E24G, i.e. the 24th amino acid, mutates from glutamic acid (E) to glycine (G), and the same applies to the others.

[0008] Furthermore, a mutant of L-rhamnose kinase with improved enzyme activity or thermal stability, wherein the amino acid sequence of the mutant is obtained by subjecting SEQ ID NO.1 to any of the following mutations:

[0009] E24G, A59W, N65L, or Q147H.

[0010] Preferably, the gene sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0011] A gene encoding the mutant.

[0012] The mutant-related biological material is any one or more combinations of the following biological materials:

[0013] (a) an expression cassette containing the above-mentioned coding gene;

[0014] (b) a recombinant expression vector containing the above encoding gene;

[0015] (c) a recombinant expression vector containing the expression cassette described in (a);

[0016] (d) a recombinant microorganism containing the above encoding gene;

[0017] (e) a recombinant microorganism containing the expression cassette described in (a);

[0018] (f) A recombinant microorganism containing the recombinant expression vector described in (b) or (c).

[0019] Furthermore, the starting vector of the recombinant expression vector in (b) and (c) is a pET series vector or a pPICZα vector, etc.; preferably a pET-28a(+) vector or a pPICZαA vector.

[0020] Furthermore, the host microorganism corresponding to the recombinant microorganism in (d), (e), and (f) is selected from prokaryotes or yeast, etc.; the prokaryotes include Escherichia ( Escherichia ) and other bacteria; the yeast includes yeast such as Pichia pastoris. More specifically, the prokaryotic organism is Escherichia coli ( Escherichia coli , E. coli ), specifically Escherichia coli BL21 (DE3); the yeast is Pichia pastoris X33 or GS115.

[0021] An immobilized enzyme comprising the mutant.

[0022] Application of the mutant, coding gene, mutant-related biological material or immobilized enzyme in the preparation of L-rhamnose kinase mutant with improved enzyme activity or thermal stability.

[0023] The use of the above mutants, encoding genes, mutant-related biomaterials or immobilized enzymes in catalyzing the phosphorylation reaction of R-type C-3 substrates; preferably, the R-type C-3 substrate includes D-psicose.

[0024] Furthermore, the mutants, encoding genes, mutant-related biomaterials or immobilized enzymes are used in the synthesis of rare sugars.

[0025] A method for obtaining the above mutant comprises the following steps: performing site-directed mutagenesis on the gene encoding the amino acid sequence of L-rhamnose kinase as shown in SEQ ID NO.1 by designing primers containing mutation sites and then expressing the site-directed mutagenesis to obtain the L-rhamnose kinase mutant.

[0026] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding the L-rhamnose kinase whose amino acid sequence is shown in SEQ ID NO.1. After correct sequencing, the gene was transformed into Escherichia coli BL21 (DE3) for expression to obtain an L-rhamnose kinase mutant with improved enzyme activity or thermal stability.

[0027] Furthermore, using plasmid pET-28a(+)-RhaB as a template, primers containing mutation sites were designed to introduce mutations into the gene encoding the L-rhamnose kinase mutant, and then transformed into Escherichia coli Top10, and transformants were randomly selected to extract plasmids for sequencing.

[0028] A method for determining the enzyme activity of an L-rhamnose glycokinase mutant, comprising the following steps:

[0029] The enzyme activity of the L-rhamnose kinase mutant was determined by liquid chromatography using D-psicose as the substrate. 5 g / L D-psicose, 5 mmol / L Mg 2+ , 30mmol / L ATP, 0.2 g / L RhaB, reacted at 37℃ for 30 min, and then heated in a 100℃ metal bath for 10 min to terminate the reaction. Centrifuged at 15000g for 10 min, the supernatant was filtered through a 0.22 µm water membrane to remove impurities, and the enzyme activity was defined by the reduction of D-psicose in the reaction using high performance liquid chromatography.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] The present invention obtains 4 mutants with improved enzyme activity or thermal stability through semi-rational design methods such as FireProt website prediction; among them, the enzyme activity of mutant N65L is 1.15 times that of the wild type. In addition, after incubation at 50°C for a certain period of time, the residual enzyme activities of mutants A59W and Q147H are 1.54 and 1.33 times that of the wild type, respectively; after incubation at 70°C for a certain period of time, the residual enzyme activities of E24G, A59W and Q147H are 2.25, 1.83 and 1.60 times that of the wild type, respectively. The present invention obtains a mutant of L-rhamnose gum sugar kinase with improved enzyme activity and thermal stability, which provides an effective way to improve the efficiency of catalyzing the synthesis of rare sugars, and has great application potential and economic value in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The relative enzyme activities of L-rhamnosyl glycokinase mutants and WT are compared.

[0033] Figure 2 Comparison of the relative residual activities of L-rhamnose kinase mutants and WT after incubation at 50 ℃.

[0034] Figure 3 Comparison of the relative residual activities of L-rhamnose kinase mutants and WT after incubation at 70 ℃. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. The operating steps or conditions not described in detail in the following embodiments are all implemented according to conventional techniques and conditions in the art. In addition, all other embodiments obtained by ordinary technicians in the art without creative work are within the scope of protection of the present invention.

[0036] In the embodiment, the L-rhamnokinase used is derived from Shigella flexneri ( Shigella flexneri ) strain, the nucleotide sequence of the coding gene after Escherichia coli codon optimization is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1, which were synthesized by a commercial company.

[0037] Example 1 Construction of single-point mutants of L-rhamnosaurus glycokinase

[0038] 1. Semi-rational design to obtain single-point mutants

[0039] The amino acid sequence of L-rhamnose kinase was submitted to the FireProt website to predict the change in binding free energy before and after the mutation of L-rhamnose kinase (ΔΔG = ΔG mutant − ΔG wild type). When ΔΔG is greater than 0, it means that the mutation makes the binding more unstable and the reaction is less likely to occur; when ΔΔG is less than 0, it means that the mutation makes the binding more stable and the reaction is more likely to occur. Therefore, we selected the following mutants with binding free energy ΔΔG less than 0 (a total of 14) as mutants to be verified: E24G, A59W, N65L, E70L, Q147H, H156W, S195L, K204P, Q244R, T275M, A279L, A287M, I340L, C346I.

[0040] 2. Construction of single-point mutants of L-rhamnosyl glycokinase

[0041] (1) Recombinant plasmid cloning

[0042] The wild-type strain recombinant plasmid pET-28a(+)-RhaB nucleotide sequence was used as a template, and the whole plasmid PCR polymerase chain reaction was performed with primers containing mutation sites. The PCR reaction system was 2×KOD buffer 25 μL, 2 mmdNTP 10 μL, 1.5 μL of upstream and downstream primers, 50-100 ng of plasmid template, 1 μL of KOD enzyme, and ddH2O was added to the final system of 50 μL. The reaction conditions were 94 ℃ 5 min; 98 ℃ 10 s, 55 ℃ 30 s, 68 ℃, 3 min 25 s, 30 cycles; 68 ℃ 10 min; stored at 4 ℃.

[0043] The recombinant plasmid pET-28a(+)-RhaB was constructed by designing homologous arm recombination primers on the pET-28a(+) vector and the nucleotide sequence (SEQ ID NO.2) of the gene encoding L-rhamnose kinase RhaB (SEQ ID NO.1) through a homologous recombination method.

[0044] The primers on the pET-28a(+) vector are:

[0045] The upstream primer was: 5′-TGAGATCCGGCTGCTAACAA-3′;

[0046] The downstream primer was: 5′-GTGATGATGATGATGATGGCT-3′;

[0047] The primers on the nucleotide sequence of the gene encoding L-rhamnokinase are:

[0048] The upstream primer was: 5′-AGCCATCATCATCATCATCACAGCAGCGGCCATATGACCTTTCGCAACT-3′.

[0049] The downstream primer was: 5′-TTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGGTGCTCGAGGGCACACA-3′.

[0050] Mutant primers are listed in Table 1.

[0051] Table 1 Mutant primers (lowercase indicates replaced codons):

[0052]

[0053] (2) Product recovery and template digestion

[0054] Product recovery: Add 50 μL of sterile water to the above PCR product, then add 200 μL of Buffer GDP, mix well, and centrifuge at 12,000 rpm for 30 s; discard the filtrate, wash twice with 700 μL of Buffer PW2 (diluted with anhydrous ethanol); finally elute with 30 μL of water and measure the concentration.

[0055] Template digestion: Add restriction endonuclease to the recovered product Dpn I, remove the template plasmid. The digestion reaction is 500-800 ng of PCR recovery product, 10× buffer 1 μL, Dpn I digestion enzyme 0.5 μL, ddH2O was added to the final system of 10 μL. The reaction conditions were 37 ℃, 30 min.

[0056] (3) Recombinant plasmid transformation and verification

[0057] Recombinant plasmid transformation: 10 μL of the above digested product was transformed into 80 μL of E. coli Top10 competent cells, spread on LB solid plates containing a final concentration of 50 μg / mL kanamycin, and cultured overnight in a 37 ℃ constant temperature incubator for 12 to 16 h.

[0058] Verification of transformed clones: Randomly pick 1 to 2 monoclonal colonies for sequencing verification.

[0059] 3. Fermentation and induced expression of single point mutants

[0060] Take 5 μL of the plasmid after successful sequencing verification and transform it into 100 μL of Escherichia coli BL21 (DE3) competent cells, spread it on an LB solid plate containing a final concentration of 50 μg / mL kanamycin, and culture it overnight in a 37°C constant temperature incubator for 12 to 16 hours.

[0061] Pick the colonies, and after verification by colony PCR, inoculate them into LB medium containing a final concentration of 50 μg / mL kanamycin, with a liquid volume of 10 mL / 50 mL, and culture them at 37 °C, 200 rpm for 12-16 h to obtain seed solution.

[0062] Initial OD 600 =0.1, transfer the seed solution to LB medium containing a final concentration of 50 μg / mL kanamycin, with a volume of 100 mL / 500 mL, and culture at 37°C with a shaker at 200 rpm until OD 600 =0.6~0.8, add IPTG with a final concentration of 0.04 mmol / L, and culture in a shaking incubator at 16 ℃ and 200 rpm for 16 h to induce expression.

[0063] Take the fermentation broth of the same OD, centrifuge at 6000 rpm for 5 min at 4 ℃, wash the bacteria once with 15 mL Tris-HCl buffer at pH7.5, and resuspend the bacteria with 20 mL Tris-HCl buffer at pH7.5. In an ice water bath, use an ultrasonic cell disruptor to disrupt the cells for 45 min (working 2 s, interval 3 s), centrifuge at 10000 rpm for 30 min at 4 ℃, and filter with a 0.22 μm filter membrane to obtain the fermentation supernatant (i.e., crude enzyme solution) of each mutant. Note: All mutants were activated, transferred, and induced under the same conditions, and the conditions for collecting and disrupting the bacteria were consistent.

[0064] Example 2 Screening of L-rhamnose glycokinase activity-enhanced mutants

[0065] For the above 14 single-point mutants, the enzyme activity of their fermentation supernatant was measured and compared with the wild type, and then single-point mutants with improved enzyme activity were screened. Finally, a single-point mutant with improved enzyme activity was obtained ( Figure 1 ).

[0066] The enzyme activity of the L-rhamnose kinase mutant was determined by liquid chromatography using D-psicose as the substrate. 5 g / L D-psicose, 5 mmol / L Mg 2+, 30mmol / L ATP, 0.2 g / L RhaB, reacted at 37℃ for 30 min, and then heated in a 100℃ metal bath for 10 min to terminate the reaction. Centrifuged at 15000g for 10 min, the supernatant was filtered through a 0.22 µm water membrane to remove impurities, and the reduction of D-psicose in the reaction was detected by high performance liquid chromatography to determine the enzyme activity of L-rhamnose kinase and enzyme mutants.

[0067] The results are as follows Figure 1 As shown in the figure, the enzyme activity of the N65L mutant is better than that of the wild type WT, which is 1.15 times that of the wild type. The activities of the other mutants were mostly not improved, and the enzyme activities were close to or lower than that of the WT.

[0068] Example 3 Screening of thermotolerant mutants of L-rhamnose kinase

[0069] For the above 14 mutants, the residual activity of their fermentation supernatants after incubation at 50 ℃ and 70 ℃ for 1 h was measured, and the wild type was treated in the same way for comparison, and then mutants with improved thermal stability were screened. Finally, 3 mutants with improved thermal stability were obtained. Figure 2 As shown in Figure 2, after being treated at 50 °C for 1 h, A59W and Q147H still maintained relatively high relative residual activities, which were 1.54 and 1.33 times that of the wild type, respectively. Figure 3 As shown in the figure, after treatment at 70 °C for 1 h, E24G, A59W and Q147H maintained high relative residual activities, which were 2.25, 1.83 and 1.60 times that of the wild type, respectively. The above results show that through the evaluation of mutant thermal stability, a total of 3 heat-resistant mutants were obtained, and their stability at high temperature was improved to a certain extent, namely E24G, A59W and Q147H.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A L-rhamnose kinase mutant, characterized in that: The amino acid sequence of the mutant is obtained by mutating SEQ ID NO.1 to A59W.

2. A gene encoding the L-rhamnokinase mutant according to claim 1.

3. The biological material related to the L-rhamnose kinase mutant according to claim 1, characterized in that: Any one or more combinations of the following biological materials: (a) an expression cassette containing the gene according to claim 2; (b) a recombinant expression vector containing the gene according to claim 2; (c) a recombinant expression vector containing the expression cassette described in (a); (d) a recombinant microorganism containing the gene according to claim 2; (e) a recombinant microorganism containing the expression cassette described in (a); (f) a recombinant microorganism containing the recombinant expression vector described in (b) or (c); The starting vector of the recombinant expression vector in (b) and (c) is a pET series vector or a pPICZα vector; The host microorganism corresponding to the recombinant microorganisms in (d), (e) and (f) is selected from Escherichia coli or Pichia pastoris.

4. An immobilized enzyme, characterized in that: Contains the L-rhamnose kinase mutant according to claim 1.

5. Use of the gene according to claim 2 or the biological material according to claim 3 in preparing a mutant of L-rhamnose kinase with improved thermal stability.

6. Use of the L-rhamnose kinase mutant of claim 1, the gene of claim 2, the biomaterial of claim 3 or the immobilized enzyme of claim 4 in catalyzing the phosphorylation reaction of D-psicose.

7. A method for obtaining the L-rhamnokinase mutant according to claim 1, characterized in that: The method comprises the following steps: performing site-directed mutagenesis on the gene encoding the amino acid sequence of L-rhamnose kinase as shown in SEQ ID NO.1 by designing primers containing mutation sites, and then expressing the site-directed mutagenesis to obtain the L-rhamnose kinase mutant according to claim 1.

8. The method according to claim 7, characterized in that: Primers containing mutation sites are designed to introduce mutations into the gene encoding the L-rhamnose kinase having an amino acid sequence as shown in SEQ ID NO.1, and after correct sequencing, the gene is transformed into Escherichia coli BL21 (DE3) for expression to obtain the L-rhamnose kinase mutant according to claim 1.

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