D-ribose-5-phosphate isomerase mutant and use thereof

Abstract

The present application relates to a kind of D-ribose-5-phosphate isomerase mutant and its application.The mutant is the 109th arginine and the 132th arginine of D-ribose-5-phosphate isomerase simultaneously occur site-directed mutation, or the 39th serine and the 109th arginine simultaneously occur site-directed mutation.Among them, the amino acid sequence of mutant R109W / R132Q is as shown in SEQ ID NO.4, and the nucleotide sequence is as shown in SEQ ID NO.3;The amino acid sequence of mutant S39I / R109F is as shown in SEQ ID NO.6, and the nucleotide sequence is as shown in SEQ ID NO.5;The amino acid sequence of mutant S39V / R109F is as shown in SEQ ID NO.8, and the nucleotide sequence is as shown in SEQ ID NO.7.Compared with wild-type D-ribose-5-phosphate isomerase, the enzyme activity of the D-ribose-5-phosphate isomerase mutant provided in the present application is higher, the efficiency of catalyzing D-allose is higher, the production cycle of D-allose is effectively shortened, and the production cost of D-allose is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a D-ribose-5-phosphate isomerase mutant and its application, and belongs to the technical field of microbial enzymes. BACKGROUND

[0002] D-allose is an aldose isomer of D-psicose, and is a C3 epimer of D-glucose. D-allose is a rare sugar with white crystal appearance, and is non-toxic, low in calories and has a wide range of physiological functions. In recent years, D-allose has become a research hotspot of functional rare sugars. The physiological functions of D-allose include low-calorie carbohydrate sweeteners and bulking agents, anticancer agents for various cancer cells, antioxidants, anti-aging, antihypertensive, cryoprotective agents and immunosuppressive agents, and have important roles in medicine and food.

[0003] The synthesis of allose includes chemical synthesis and biological synthesis. The chemical synthesis method is to synthesize allose by changing the configuration of various sugars through redox pathways, but the process is complicated and has many by-products. The biological transformation method includes isomerization of allose by isomerase to generate allose, and two-step enzyme reaction catalysis from D-fructose, in which D-fructose is first catalyzed by D-tagatose-3-epimerase to generate D-allose. The rare sugar D-allose is then converted to D-allose by ketone aldehyde isomerase. It is reported that ketone aldehyde isomerase for the synthesis of D-allose mainly includes L-rhamnose isomerase, D-ribose-5-phosphate isomerase and D-galactose-6-phosphate isomerase. Compared with chemical methods, biological synthesis methods have many advantages, such as easier processing, higher activity, stronger specificity under mild conditions and lower by-product conversion rate.

[0004] D-ribose-5-phosphate isomerase is widely present in various microorganisms, and the enzyme can be used to isomerize D-allose to aldose to synthesize D-allose. However, a by-product altritol is easily produced in the conversion process. Compared with chemical methods, the current biological synthesis enzyme method is more convenient, but the existing D-ribose-5-phosphate isomerase has a low conversion rate. There is no report on D-ribose-5-phosphate isomerase with improved catalytic activity, so it is of great significance to use directed evolution technology to modify D-ribose-5-phosphate isomerase, which can provide important technical support for industrial production and application. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a D-ribose-5-phosphate isomerase mutant and its application. The present application uses site-directed mutagenesis technology to direct evolution of wild-type D-ribose-5-phosphate isomerase, and obtains a mutant enzyme with higher efficiency in catalyzing the conversion of D-ribulose into D-allose, which is used in the enzyme method large-scale industrial production of D-allose, so as to replace the chemical method and other enzyme methods which are complicated and have many by-products to produce D-allose with many physiological functions.

[0006] The technical scheme of the present application is as follows:

[0007] A D-ribose-5-phosphate isomerase mutant, wherein the mutant is a D-ribose-5-phosphate isomerase with simultaneous site mutations of arginine at position 109 and arginine at position 132, or simultaneous site mutations of serine at position 39 and arginine at position 109; the amino acid sequence of the D-ribose-5-phosphate isomerase is shown as SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.

[0008] According to the present application, preferably, the mutant is a D-ribose-5-phosphate isomerase mutant R109W / R132Q, the amino acid sequence of which is shown as SEQ ID NO. 4, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 3; which is a D-ribose-5-phosphate isomerase with simultaneous mutations of arginine at position 109 into tryptophan and arginine at position 132 into glutamine.

[0009] According to the present application, preferably, the mutant is a D-ribose-5-phosphate isomerase mutant S39I / R109F, the amino acid sequence of which is shown as SEQ ID NO. 6, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 5; which is a D-ribose-5-phosphate isomerase with simultaneous mutations of serine at position 39 into isoleucine and arginine at position 109 into phenylalanine.

[0010] According to the present application, preferably, the mutant is a D-ribose-5-phosphate isomerase mutant S39V / R109F, the amino acid sequence of which is shown as SEQ ID NO. 8, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 7; which is a D-ribose-5-phosphate isomerase with simultaneous mutations of serine at position 39 into valine and arginine at position 109 into phenylalanine.

[0011] A recombinant vector, which is a coding gene of the above-mentioned D-ribose-5-phosphate isomerase mutant inserted in a plasmid vector.

[0012] According to the present application, preferably, the plasmid vector is pET-21a(+).

[0013] A recombinant strain is obtained by transforming the above recombinant vector into a host cell.

[0014] According to the present invention, preferably, the host cell is Escherichia coli; further preferably, the host cell is Escherichia coli BL21.

[0015] Application of the D-ribose-5-phosphate isomerase mutant in the preparation of D-allose.

[0016] According to the preferred embodiment of the present invention, the preparation process in the application is as follows:

[0017] (1) adding D-psicose to 0.01-0.03 mM glycine-sodium hydroxide to obtain a D-psicose solution;

[0018] (2) Preheat the D-psicose solution at 30-80°C for 0.5-1.5 min, then add the D-ribose-5-phosphate isomerase mutant enzyme solution and Mn 2+ solution to obtain an enzyme reaction system;

[0019] (3) The enzyme reaction system was reacted at 30-80° C. for 15-25 hours. After the reaction was completed, the solution was boiled for 10 minutes, the supernatant was collected by centrifugation, diluted, and then subjected to membrane separation to obtain D-allose.

[0020] According to the preferred embodiment of the present invention, in step (2), the concentration of D-psicose in the enzyme reaction system is 90-110 g / L, the concentration of D-ribose-5-phosphate isomerase mutant is 0.1-0.3 mg / mL, and Mn 2+ The concentration is 0.5~1.5mM.

[0021] The beneficial effects of the present invention are:

[0022] The present invention is based on D-ribose-5-phosphate isomerase, and the amino acids at positions 39, 109, and 132 thereof are selected for site-directed mutagenesis. At the same time, the arginine at position 109 is mutated to tryptophan and the arginine at position 132 is mutated to glutamine to form a double mutant, thereby obtaining the D-ribose-5-phosphate isomerase mutant R109W / R132Q; at the same time, the serine at position 39 is mutated to isoleucine and the arginine at position 109 is mutated to phenylalanine to form a double mutant, thereby obtaining the D-ribose-5-phosphate isomerase mutant S39I / R109F; at the same time, the serine at position 39 is mutated to valine and the arginine at position 109 is mutated to phenylalanine to form a double mutant, thereby obtaining the D-ribose-5-phosphate isomerase mutant S39V / R109F. The D-ribose-5-phosphate isomerase mutant provided by the present invention has higher enzymatic activity and catalytic efficiency of D-allose. Compared with wild-type D-ribose-5-phosphate isomerase, the enzyme activity of the D-ribose-5-phosphate isomerase mutant R109W / R132Q increased by 1.39 times at 40°C, and the conversion rate increased by 5.99%. At 60°C, the enzyme activity of the D-ribose-5-phosphate isomerase mutant S39I / R109F increased by 1.58 times, and the conversion rate increased by 9.87%. At 80°C, the enzyme activity of the D-ribose-5-phosphate isomerase mutant S39V / R109F increased by 1.9 times, and the conversion rate increased by 14.7%. This effectively shortens the production cycle of D-allose and reduces the production cost of D-allose, thus solving the problems of low D-ribose-5-phosphate isomerase activity, low expression level, limited application, and low D-allose synthesis yield in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis results of D-ribose-5-phosphate isomerase mutants are shown;

[0024] Among them, lane M is a protein marker, lane 1 is the D-ribose-5-phosphate isomerase mutant R109W / R132Q, lane 2 is the D-ribose-5-phosphate isomerase mutant S39I / R109F, and lane 3 is the D-ribose-5-phosphate isomerase mutant S39V / R109F.

[0025] Figure 2 Schematic diagram of the synthesis of D-allose using D-ribose-5-phosphate isomerase mutants.

[0026] Figure 3 is the relative enzyme activity of D-ribose-5-phosphate isomerase mutant R109W / R132Q at 40°C.

[0027] Figure 4 is the relative enzyme activity of D-ribose-5-phosphate isomerase mutant S39I / R109F at 60°C.

[0028] Figure 5 is the relative enzyme activity of D-ribose-5-phosphate isomerase mutant S39V / R109F at 80°C. DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are further described below in conjunction with the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0030] Example 1. Preparation of D-ribose-5-phosphate isomerase mutants

[0031] 1. The inventors conducted biological analysis on the amino acid sequence (SEQ ID NO. 2) and nucleotide sequence (SEQ ID NO. 1) of the wild-type D-ribose-5-phosphate isomerase from Acetivibrio thermocellum.

[0032] Specifically, the researchers used ESPript software to analyze highly conserved regions in the amino acid sequence. They then used the Swiss-Model tool to perform protein modeling of the wild-type D-ribose 5-phosphate isomerase. PyMol and DiscoverStudio software were used to predict the enzyme's active site. They found that amino acid positions 39, 109, and 132 of the wild-type D-ribose 5-phosphate isomerase sequence are located near the active center and highly conserved regions, suggesting that site-directed mutagenesis of these positions is highly likely to improve the enzyme's catalytic activity towards D-psicose. Therefore, single and double site-directed mutagenesis were performed at positions 39, 109, and 132 of the wild-type D-ribose 5-phosphate isomerase sequence. After screening, the D-ribose 5-phosphate isomerase mutants R109W / R132Q, S39I / R109F, and S39V / R109F were obtained.

[0033] The amino acid sequence of the D-ribose-5-phosphate isomerase mutant R109W / R132Q is shown in SEQ ID NO. 4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 3. The amino acid sequence of the D-ribose-5-phosphate isomerase mutant S39I / R109F is shown in SEQ ID NO. 6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 5. The amino acid sequence of the D-ribose-5-phosphate isomerase mutant S39V / R109F is shown in SEQ ID NO. 8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 7.

[0034] 2. Heterologous expression of D-ribose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F, and S39V / R109F (1) Using the D-ribose-5-phosphate isomerase mutant R109W / R132Q as an example, single-point mutation primers R109F-F / R and R132Q-F / R were designed. Then, using the nucleotide sequence of wild-type D-ribose-5-phosphate isomerase as a template, double-site mutagenesis was performed by inverse PCR amplification using primers R109F-F / R and R132Q-F / R. After the amplification, the unmutated original template was removed with DpnⅠ enzyme to obtain the mutant R109W / R132Q gene as shown in SEQ ID NO. 3.

[0035] The primer sequences are:

[0036] R109F-F: 5'-GGGCGAGtttGTTGTTGGTCTGGACCTGGCTT-3',

[0037] R109F-R: 5'-CAACAACaaaCTCGCCCAACGGCCAGAATGTTC-3';

[0038] R132Q-F: 5'-ATTTCAGGGTGGCcagCACGCTACCCGTGTGGGC-3',

[0039] R132Q-R: 5'-ctgGCCACCCTGAAATTCCGCCTTCAGCCAGG-3';

[0040] (2) The mutant R109W / R132Q gene was ligated to pET21a(+) to obtain the recombinant plasmid pET21a(+)-Rpi-R109W / R132Q; the recombinant plasmid pET21a(+)-Rpi-R109W / R132Q was then transformed into Escherichia coli BL21 competent cells to construct the recombinant strain Rpi-R109W / R132Q;

[0041] (3) The recombinant strain Rpi-R109W / R132Q was cultured at 37°C and 200 rpm with shaking until the OD was 0.6-0.8. IPTG (final concentration 0.2 mM) was added and induced for 20 h. The cells were collected by centrifugation and resuspended in PBS buffer. The cells were ultrasonically disrupted and centrifuged. The supernatant was retained and purified and eluted using a nickel column to obtain the heterologously expressed D-ribose-5-phosphate isomerase mutant R109W / R132Q.

[0042] The D-ribose-5-phosphate isomerase mutants S39I / R109F and S39V / R109F were obtained by heterologous expression and purification according to the same method as above. The primer sequences involved are:

[0043] S39I-F: 5'-GAAattGTCGACTACCCGGACTTTGGCCTCAA-3',

[0044] S39I-R: 5'-GGGTAGTCGACaatTTCATTACCATGGGTGCCGA-3';

[0045] R109F-F: 5'-GGGCGAGtttGTTGTTGGTCTGGACCTGGCTT-3',

[0046] R109F-R: 5'-CAACAACaaaCTCGCCCAACGGCCAGAATGTTC-3';

[0047] S39V-F: 5'-GTAATGAAgtgGTCGACTACCCGGACTTTGGC-3',

[0048] S39V-R: 5'-GTCGACcacTTCATTACCATGGGTGCCGAAAT-3';

[0049] R109F-F: 5'-GGGCGAGtttGTTGTTGGTCTGGACCTGGCTT-3',

[0050] R109F-R: 5'-CAACAACaaaCTCGCCCAACGGCCAGAATGTTC-3';

[0051] The PCR amplification system was prepared according to the kit instructions; the PCR amplification procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec; annealing at 60°C for 30 sec; extension at 72°C for 3 min 20 sec (30 cycles); termination of extension at 72°C for 10 min; and finally, incubation at 4°C.

[0052] The expression of the purified recombinant proteins R109W / R132Q, S39I / R109F and S39V / R109F was identified by polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 shown.

[0053] Depend on Figure 1It can be seen that the band sizes of D-ribose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F and S39V / R109F are all around 16.5 kDa, which is consistent with the predicted value, indicating that D-ribose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F and S39V / R109F were successfully heterologously expressed.

[0054] Example 2: Preparation of D-allose using D-ribose-5-phosphate isomerase mutant

[0055] The nucleotide sequence of wild-type D-ribose-5-phosphate isomerase was artificially synthesized, and the wild-type D-ribose-5-phosphate isomerase was obtained according to the heterologous expression method described in Example 1.

[0056] A method for producing D-allose using D-psicose as a substrate comprises the following steps:

[0057] (1) Adding D-psicose to 0.02 mM glycine-sodium hydroxide (pH = 9.0) to obtain a D-psicose solution;

[0058] (2) 1 mL of D-psicose solution was preheated at 40°C, 60°C, and 80°C for 1 min, and then 1 mL of wild-type D-ribose-5-phosphate isomerase enzyme solution and 1 mL of Mn 2+ solution to obtain an enzyme reaction system;

[0059] In the enzyme reaction system, the concentration of D-psicose was 100 g / L, the concentration of D-ribose-5-phosphate isomerase mutant was 0.2 mg / mL, and the concentration of Mn 2+ The concentration is 1mM;

[0060] (3) The enzyme reaction system was reacted at 40°C, 60°C, and 80°C for 20 h, respectively. After the reaction, the reaction was boiled for 10 min, and the supernatant was collected by centrifugation. After dilution 10 times, membrane separation was performed to obtain D-allose. The yields of D-allose measured by HPLC were 32.3 g / L, 25.11 g / L, and 17.09 g / L, respectively. The principle of synthesizing D-allose using D-ribose-5-phosphate isomerase is as follows Figure 2 shown.

[0061] The same method was used to determine the yield of D-allose produced by using the D-ribose-5-phosphate isomerase mutants R109W / R132Q, D-ribose-5-phosphate isomerase mutants S39I / R109F, and D-ribose-5-phosphate isomerase mutants S39V / R109F.

[0062] The determination results are: the D-allose yield of mutant R109W / R132Q is 38.29 g / L at 40℃, the D-allose yield of mutant S39I / R109F is 34.98 g / L at 60℃, and the D-allose yield of mutant S39V / R109F is 31.79 g / L at 80℃.

[0063] According to the above data, at 40℃, the efficiency of wild-type D-ribulose-5-phosphate isomerase in catalyzing the conversion of D-psicose into D-allose is 32.3%, and the efficiency of D-ribulose-5-phosphate isomerase mutant R109W / R132Q in catalyzing the conversion of D-psicose into D-allose is 38.29%; at 60℃, the efficiency of wild-type D-ribulose-5-phosphate isomerase in catalyzing the conversion of D-psicose into D-allose is 25.11%, and the efficiency of D-ribulose-5-phosphate isomerase mutant S39I / R109F in catalyzing the conversion of D-psicose into D-allose is 34.98%; at 80℃, the efficiency of wild-type D-ribulose-5-phosphate isomerase in catalyzing the conversion of D-psicose into D-allose is 17.09%, and the efficiency of D-ribulose-5-phosphate isomerase mutant S39V / R109F in catalyzing the conversion of D-psicose into D-allose is 31.79%. Compared with wild-type D-ribulose-5-phosphate isomerase, the conversion rates of D-ribulose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F and S39V / R109F are increased by 5.99%, 9.87% and 14.7%, respectively. The production cycle of D-allose can be effectively shortened, and the production cost of D-allose can be reduced, thereby solving the problems of low activity, low expression and limited application of D-ribulose-5-phosphate isomerase in the prior art, and low yield of D-allose in synthesis.

[0064] The calculation formula of conversion rate is: conversion rate = concentration of product D-allose / concentration of substrate D-psicose.

[0065] Example 3, Effect of Different Temperatures on Enzyme Activity of D-ribulose-5-phosphate Isomerase Mutants

[0066] According to the method described in Example 2, the enzyme activity of D-ribulose-5-phosphate isomerase double mutants R109W / R132Q, S39I / R109F and S39V / R109F in catalyzing the conversion of D-psicose into D-allose at 40℃, 60℃ and 80℃, respectively, was detected. The enzyme activity of wild-type D-ribulose-5-phosphate isomerase in producing D-allose was taken as the relative enzyme activity of 100%, and the relative enzyme activity of each D-ribulose-5-phosphate isomerase mutant at 40℃ is shown in Table 1. Figure 3 The relative enzyme activity of each D-ribulose-5-phosphate isomerase mutant at 60℃ is shown in Table 2. Figure 4The relative enzyme activities of the D-ribose-5-phosphate isomerase mutants at 80°C are shown in Figure 5 shown.

[0067] Depend on Figures 3 to 5 It can be seen that compared with the wild-type D-ribose-5-phosphate isomerase, at 40°C, the enzyme activity of the D-ribose-5-phosphate isomerase mutant R109W / R109Q increased by 1.39 times, the D-ribose-5-phosphate isomerase mutant S39I / R109F increased by 1.58 times, and the D-ribose-5-phosphate isomerase mutant S39V / R109F increased by 1.9 times. This shows that the D-ribose-5-phosphate isomerase mutants of the present invention can greatly improve the catalytic efficiency, among which the D-ribose-5-phosphate isomerase mutant S39V / R109F has the best effect in catalyzing the conversion of D-psicose to D-allose.

Claims

1. A D-ribose-5-phosphate isomerase mutant, characterized in that The amino acid sequence of the D-ribose-5-phosphate isomerase mutant is SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.

8.

2. The gene encoding the D-ribose-5-phosphate isomerase mutant according to claim 1, characterized in that: The nucleotide sequence of the coding gene is SEQ ID NO.3, SEQ ID NO.5 or SEQ ID NO.

7.

3. A recombinant vector, characterized in that The gene encoding the D-ribose-5-phosphate isomerase mutant according to claim 2 is inserted into a plasmid vector.

4. The recombinant vector according to claim 3, wherein The plasmid vector is pET-21a (+).

5. A recombinant strain, characterized in that The method is obtained by transforming the recombinant vector according to claim 3 into a host cell.

6. The recombinant strain according to claim 5, characterized in that The host cell is Escherichia coli.

7. The recombinant strain according to claim 6, characterized in that The host cell is Escherichia coli BL21.

8. Use of the D-ribose-5-phosphate isomerase mutant according to claim 1 in the preparation of D-allose.

9. The use according to claim 8, characterized in that The preparation process in the application is as follows: (1) Adding D-psicose to 0.01-0.03 mM glycine-sodium hydroxide to obtain a D-psicose solution; (2) Preheat the D-psicose solution at 30-80°C for 0.5-1.5 min, then add the D-ribose-5-phosphate isomerase mutant enzyme solution and Mn 2+ solution to obtain an enzyme reaction system; (3) The enzyme reaction system was reacted at 30-80°C for 15-25 hours. After the reaction, the solution was boiled for 10 minutes, and the supernatant was collected by centrifugation. The supernatant was diluted and then subjected to membrane separation to obtain D-allose.

10. The use according to claim 9, characterized in that In step (2), the concentration of D-psicose in the enzyme reaction system is 90-110 g / L, the concentration of D-ribose-5-phosphate isomerase mutant is 0.1-0.3 mg / mL, and the concentration of Mn 2+ The concentration is 0.5~1.5mM.

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