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

Abstract

The invention relates to a D-ribose-5-phosphate isomerase mutant and an application of the D-ribose-5-phosphate isomerase mutant. According to the mutant, site-specific mutagenesis is carried out on 109th arginine and 132th arginine of D-ribose-5-phosphate isomerase at the same time, or site-specific mutagenesis is carried out on 39th serine and 109th arginine of D-ribose-5-phosphate isomerase at the same time. Wherein the amino acid sequence of the mutant R109W / R132Q is as shown in SEQ ID NO.4, and the nucleotide sequence of the mutant R109W / R132Q is as shown in SEQ ID NO.3; the amino acid sequence of the mutant S39I / R109F is as shown in SEQ ID NO.6, and the nucleotide sequence of the mutant S39I / R109F is as shown in SEQ ID NO.5; the amino acid sequence of the mutant S39V / R109F is as shown in SEQ ID NO.8, and the nucleotide sequence of the mutant S39V / R109F is as shown in SEQ ID NO.7. Compared with wild type D-ribose-5-phosphate isomerase, the D-ribose-5-phosphate isomerase mutant provided by the invention has the advantages that the enzyme activity and the efficiency of catalyzing D-allose are higher, the production period of the D-allose is effectively shortened, and the production cost of the D-allose is reduced.

Description

Technical Field

[0001] The invention relates to a D-ribose-5-phosphate isomerase mutant and application thereof, belonging to the technical field of microbial enzymes. Background Art

[0002] D-allose is the aldose isomer of D-psicose and is a C3 diastereomer of D-glucose. D-allose is a rare sugar with a wide range of physiological functions and is a white crystal. In recent years, it has become a research hotspot for functional rare sugars. The physiological functions of D-allose include being a low-calorie carbohydrate sweetener and bulking agent, an anticancer agent for various cancer cells, an antioxidant, anti-aging, anti-hypertension, cryoprotectant and immunosuppressant, etc. It plays an important role in medicine and food.

[0003] The synthesis of allose can be divided into chemical synthesis and biosynthesis. The chemical synthesis method is to synthesize allose by changing the configuration of various sugars through redox pathways, but the process is cumbersome and has many by-products. The biotransformation method includes the isomerization of psicose to allose using isomerase, and the biosynthesis from D-fructose is catalyzed by a two-step enzyme reaction, in which D-fructose is first catalyzed by D-tagatose-3-isomerase to produce D-psicose. The generated rare sugar D-psicose is then converted into D-allose by ketoaldehyde isomerase. It is reported that the ketoaldehyde isomerase used 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, biosynthesis has advantages in many aspects, such as easier processing, higher activity, stronger specificity under mild conditions, and low by-product conversion rate.

[0004] D-ribose-5-phosphate isomerase is widely present in various microorganisms. The enzyme can be used to isomerize D-psicose into aldose to synthesize D-allose. However, the byproduct altrose is very easy to be produced during the conversion process. Compared with chemical methods, the current biosynthetic enzyme method is more convenient, but the existing D-ribose-5-phosphate isomerase has a low conversion rate. There is currently no report on D-ribose-5-phosphate isomerase with improved catalytic activity. Therefore, it is of great significance to use directed evolution technology to transform D-ribose-5-phosphate isomerase, which can provide important technical support for industrial production and application. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a D-ribose-5-phosphate isomerase mutant and its application. The present invention uses site-directed mutagenesis technology to perform directed evolution on wild-type D-ribose-5-phosphate isomerase, and obtains a mutant enzyme that catalyzes the conversion of D-psicose into D-allose with higher efficiency, which is used for large-scale industrial production of D-allose by enzymatic method, thereby replacing chemical methods and other enzymatic methods with complicated processes and many by-products to produce D-allose with multiple physiological functions.

[0006] The technical solution of the present invention is as follows:

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

[0008] Preferably according to the present invention, the mutant is a D-ribose-5-phosphate isomerase mutant R109W / R132Q, whose amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the arginine at position 109 of the amino acid sequence of D-ribose-5-phosphate isomerase is mutated into tryptophan, and the arginine at position 132 is mutated into glutamine.

[0009] Preferably according to the present invention, the mutant is a D-ribose-5-phosphate isomerase mutant S39I / R109F, whose amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the serine at position 39 of the amino acid sequence of D-ribose-5-phosphate isomerase is mutated into isoleucine, and the arginine at position 109 is mutated into phenylalanine.

[0010] Preferably according to the present invention, the mutant is a D-ribose-5-phosphate isomerase mutant S39V / R109F, whose amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; the serine at position 39 of the amino acid sequence of D-ribose-5-phosphate isomerase is mutated into valine, and the arginine at position 109 is mutated into phenylalanine.

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

[0012] Preferably according to the present invention, the plasmid vector is pET-21a(+).

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

[0014] Preferably according to the present invention, 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] Preferably, according to 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) After preheating the D-psicose solution at 30-80°C for 0.5-1.5 min, 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 is reacted at 30-80° C. for 15-25 hours. After the reaction is completed, the reaction is boiled for 10 minutes, the supernatant is collected by centrifugation, and the supernatant is diluted and then subjected to membrane separation to obtain D-allose.

[0020] Preferably, 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.

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

[0022] The present invention is based on D-ribose-5-phosphate isomerase, selects the 39th, 109th and 132nd amino acids thereof for site-directed mutation, and simultaneously mutates the 109th arginine into tryptophan and the 132nd arginine into glutamine to form a double mutant, thereby obtaining a D-ribose-5-phosphate isomerase mutant R109W / R132Q; simultaneously mutates the 39th serine into isoleucine and the 109th arginine into phenylalanine to form a double mutant, thereby obtaining a D-ribose-5-phosphate isomerase mutant S39I / R109F; simultaneously mutates the 39th serine into valine and the 109th arginine into phenylalanine to form a double mutant, thereby obtaining a D-ribose-5-phosphate isomerase mutant S39V / R109F. The D-ribose-5-phosphate isomerase mutant provided by the present invention has higher enzyme activity and catalytic efficiency of D-allose. Compared with wild-type D-ribose-5-phosphate isomerase, at 40°C, the enzyme activity of D-ribose-5-phosphate isomerase mutant R109W / R132Q increased by 1.39 times, and the conversion rate increased by 5.99%; at 60°C, the enzyme activity of 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 D-ribose-5-phosphate isomerase mutant S39V / R109F increased by 1.9 times, and the conversion rate increased by 14.7%. The production cycle of D-allose is effectively shortened, and the production cost of D-allose is reduced, thereby solving the problems of low activity, low expression level, limited application, and low synthesis yield of D-allose in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[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 solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope 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 mutant

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

[0032] Specifically, the highly conserved regions of the amino acid sequence were analyzed using ESPript software, and then the wild-type D-ribose-5-phosphate isomerase was modeled using Swiss-Model, and the active center of the enzyme was predicted using pymol and Discoverstudio software. It was found that the 39th, 109th and 132nd positions of the amino acid sequence of the wild-type D-ribose-5-phosphate isomerase were located near the active center and the highly conserved region, and site-directed mutations were very likely to improve the catalytic activity of D-ribose-5-phosphate isomerase to D-psicose. Therefore, single and double site-directed mutations were performed on the 39th, 109th and 132nd positions of the amino acid sequence of the wild-type D-ribose-5-phosphate isomerase, and after screening, the D-ribose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F and S39V / R109F were obtained.

[0033] Among them, 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) Taking 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, the nucleotide sequence of wild-type D-ribose-5-phosphate isomerase was used as a template, and primers R109F-F / R and R132Q-F / R were used to perform double-site-directed mutagenesis by inverse PCR amplification. After the amplification was completed, 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 connected 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 the culture was induced for 20 h. The cells were collected by centrifugation and resuspended in PBS buffer, and ultrasonically disrupted. The disrupted cells were centrifuged and the supernatant was retained. The supernatant was then purified and eluted using a nickel column to obtain the heterologously expressed D-ribose-5-phosphate isomerase mutant R109W / R132Q.

[0042] According to the same method as above, heterologous expression and purification were performed to obtain D-ribose-5-phosphate isomerase mutants S39I / R109F and S39V / R109F. 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 instructions of the kit; the PCR amplification program was as follows: pre-denaturation, 95°C for 5 min; denaturation, 95°C for 30 sec; annealing, 60°C for 30 sec; extension, 72°C for 3 min 20 sec (30 cycles); termination of extension, 72°C for 10 min; and finally incubation at 4°C.

[0052] The expression of purified recombinant proteins R109W / R132Q, S39I / R109F and S39V / R109F was identified by 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 are 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, comprising 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) After preheating 1 mL of D-psicose solution at 40°C, 60°C, and 80°C for 1 min, 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 diluting 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 measurement results showed that the D-allose production of mutant R109W / R132Q was 38.29 g / L at 40°C, the D-allose production of mutant S39I / R109F was 34.98 g / L at 60°C, and the D-allose production of mutant S39V / R109F was 31.79 g / L at 80°C.

[0063] According to the above data, at 40°C, the efficiency of wild-type D-ribose-5-phosphate isomerase in catalyzing the conversion of D-psicose to D-allose was 32.3%, and the efficiency of D-ribose-5-phosphate isomerase mutant R109W / R132Q in catalyzing the conversion of D-psicose to D-allose was 38.29%; at 60°C, the efficiency of wild-type D-ribose-5-phosphate isomerase in catalyzing the conversion of D-psicose to D-allose was 25.1 1%, the efficiency of D-psicose converted to D-allose by D-ribose-5-phosphate isomerase mutant S39I / R109F was 34.98%; at 80°C, the efficiency of D-psicose converted to D-allose by wild-type D-ribose-5-phosphate isomerase was 17.09%, and the efficiency of D-psicose converted to D-allose by D-ribose-5-phosphate isomerase mutant S39V / R109F was 31.79%. Compared with wild-type D-ribose-5-phosphate isomerase, the conversion rates of D-ribose-5-phosphate isomerase mutants R109W / R132Q, S39I / R109F and S39V / R109F of the present invention were increased by 5.99%, 9.87% and 14.7%, respectively. The method can effectively shorten the production cycle of D-allose and reduce the production cost of D-allose, thereby solving the problems of low activity, low expression level, limited application and low synthesis yield of D-allose in the prior art.

[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 the activity of D-ribose-5-phosphate isomerase mutants

[0066] According to the method described in Example 2, the enzyme activities of the double mutants of D-ribose-5-phosphate isomerase R109W / R132Q, S39I / R109F and S39V / R109F in catalyzing the conversion of D-psicose to D-allose at 40°C, 60°C and 80°C were respectively detected. The relative enzyme activity of the wild-type D-ribose-5-phosphate isomerase to produce D-allose was taken as 100%. The relative enzyme activities of the D-ribose-5-phosphate isomerase mutants at 40°C were as follows: Figure 3 The relative enzyme activities of the D-ribose-5-phosphate isomerase mutants at 60°C are shown in 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, indicating that the D-ribose-5-phosphate isomerase mutant in 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 mutant is a D-ribose-5-phosphate isomerase in which the arginine at position 109 and the arginine at position 132 undergo site-directed mutations simultaneously, or the serine at position 39 and the arginine at position 109 undergo site-directed mutations simultaneously; the amino acid sequence of the D-ribose-5-phosphate isomerase is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

2. The D-ribose-5-phosphate isomerase mutant according to claim 1, characterized in that The mutant is a D-ribose-5-phosphate isomerase mutant R109W / R132Q, whose amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the arginine at position 109 of the D-ribose-5-phosphate isomerase amino acid sequence is mutated into tryptophan, and the arginine at position 132 is mutated into glutamine.

3. The D-ribose-5-phosphate isomerase mutant according to claim 1, characterized in that The mutant is a D-ribose-5-phosphate isomerase mutant S39I / R109F, whose amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the 39th serine in the amino acid sequence of D-ribose-5-phosphate isomerase is mutated into isoleucine, and the 109th arginine is mutated into phenylalanine.

4. The D-ribose-5-phosphate isomerase mutant according to claim 1, characterized in that The mutant is a D-ribose-5-phosphate isomerase mutant S39V / R109F, whose amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; the 39th serine in the amino acid sequence of D-ribose-5-phosphate isomerase is mutated into valine, and the 109th arginine is mutated into phenylalanine.

5. A recombinant vector, characterized in that: The gene encoding the D-ribose-5-phosphate isomerase mutant is inserted into a plasmid vector; Preferably, the plasmid vector is pET-21a(+).

6. A recombinant strain, characterized in that: The method is obtained by transforming the above recombinant vector into a host cell; Preferably, the host cell is Escherichia coli; further preferably, the host cell is Escherichia coli BL21.

7. Use of the D-ribose-5-phosphate isomerase mutant according to any one of claims 1 to 4 in the preparation of D-allose.

8. The use according to claim 7, 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) After preheating the D-psicose solution at 30-80°C for 0.5-1.5 min, 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 is reacted at 30-80° C. for 15-25 hours. After the reaction is completed, the reaction is boiled for 10 minutes, the supernatant is collected by centrifugation, and the supernatant is diluted and then subjected to membrane separation to obtain D-allose.

9. The use according to claim 8, 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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