A ribitol dehydrogenase mutant with improved substrate selectivity and its application

Abstract

The present invention discloses a ribitol dehydrogenase mutant with improved substrate selectivity and its application. The ribitol dehydrogenase mutant is obtained by targeted mutation of amino acid residues in the substrate binding site of wild-type ribitol dehydrogenase, and comprises any one of E168D, V172T, and F310W mutations or a combination thereof. The ribitol dehydrogenase mutant disclosed in the present invention can selectively recognize fructose as a substrate in a mixed substrate system of fructose and psicose, catalyze the production of sorbitol, and achieve a significant improvement in substrate selectivity and conversion rate. The present invention provides a catalyst comprising the ribitol dehydrogenase mutant with improved substrate selectivity, and the temperature and pH conditions of the catalytic reaction are easy to achieve. The present invention provides a gene encoding the ribitol dehydrogenase mutant, an expression vector comprising the gene encoding the ribitol dehydrogenase mutant, and a recombinant bacterium comprising the expression vector. The present invention also provides a preparation and application method of the ribitol dehydrogenase mutant.

Description

Technical Field

[0001] The present invention relates to a ribitol dehydrogenase mutant, in particular to a ribitol dehydrogenase mutant with improved substrate selectivity and application thereof in catalyzing the conversion of fructose into sorbitol, and belongs to the fields of genetic engineering and enzyme engineering. Background Art

[0002] Rare sugars are a class of monosaccharides and sugar alcohols that are rarely found in nature. They taste similar to sucrose but offer advantages such as low calorie count, high stability, non-hygroscopicity, and non-cariogenicity. They possess unique physiological functions and significant application value. As functional sweeteners, rare sugars are ideal sucrose substitutes for patients with diabetes and obesity. These functional sugars and sugar alcohols are of significant importance to human health, and related glycoengineering products have already played a vital role in my country's food, health products, pharmaceuticals, and agriculture sectors. With the increasing optimization of China's sugar consumption structure, the application potential of functional rare sugars is enormous.

[0003] Sorbitol is a six-carbon rare sugar alcohol that combines the advantages of polyols, such as low calories, low sugar content, and anti-caries properties. It is widely used in the food, daily chemical, and pharmaceutical industries as a sweetener, humectant, excipient, and preservative. Sorbitol is found naturally in pears, peaches, and apples, at concentrations of approximately 1% to 2%. Sorbitol can be artificially produced through hydrogenation, electrochemical methods, and fermentation. Biofermentation, for example, converts fructose into sorbitol through enzyme catalysis.

[0004] Ribitol dehydrogenases are oxidoreductases that reversibly convert D-ribulose to ribitol. Based on their coenzyme specificity, ribitol dehydrogenases can be divided into NADH-dependent and NADPH-dependent types. A biocatalytic system combining ribitol dehydrogenase and a regenerative coenzyme system can achieve the direct conversion of fructose to sorbitol. However, currently known ribitol dehydrogenases suffer from poor substrate selectivity and low catalytic efficiency, making them unsuitable for industrial production. Summary of the Invention

[0005] Objective of the invention: The present invention provides a ribitol dehydrogenase mutant. By directing the amino acid sequence of the substrate-binding site of the wild-type ribitol dehydrogenase, a ribitol dehydrogenase mutant with high substrate selectivity and high yield in the catalytic conversion of fructose to sorbitol was obtained.

[0006] Technical solution: In the first aspect, the present invention provides a ribitol dehydrogenase mutant with improved substrate selectivity, which is obtained by subjecting the amino acid sequence shown in SEQ ID NO.1 to at least one mutation among E168D, V172T, and F310W.

[0007] In a second aspect, the present invention provides a ribitol dehydrogenase gene, which encodes the ribitol dehydrogenase mutant according to the first aspect. The gene is obtained by base mutation of the nucleotide sequence shown in SEQ ID NO. 2.

[0008] In a third aspect, the present invention provides an expression vector comprising the nucleotide sequence of the second aspect, wherein the expression vector is a pET series expression vector.

[0009] In a fourth aspect, the present invention provides a recombinant bacterium, which is Escherichia coli comprising the expression vector of the third aspect.

[0010] In a fifth aspect, the present invention provides a method for constructing a recombinant bacterium, comprising the following steps: (1) constructing an expression vector: connecting the gene of the second aspect with an enzyme-cut plasmid to obtain a recombinant expression vector; (2) constructing a recombinant bacterium: transferring the constructed recombinant expression vector into competent Escherichia coli cells, culturing and screening to obtain recombinant bacteria.

[0011] In a sixth aspect, the present invention provides a catalyst comprising the enzyme mutant of the first aspect.

[0012] In a seventh aspect, the present invention provides a use of a ribitol dehydrogenase mutant. In a mixed system of fructose and psicose, the ribitol dehydrogenase mutant of the first aspect can selectively convert fructose into sorbitol. The reaction conditions include a reaction temperature of 25-45°C, a pH of 6.5-10, and a reaction time of 6-16 hours.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0014] 1. The ribitol dehydrogenase mutant obtained in the present invention can selectively recognize fructose as a substrate in a mixed substrate system of fructose and psicose, catalyze the production of sorbitol, and achieve significant improvements in substrate selectivity and conversion rate.

[0015] 2. The present invention provides a catalyst comprising a ribitol dehydrogenase mutant with improved substrate selectivity. The temperature and pH conditions of the catalytic reaction are easy to achieve and have a basis for industrial production application.

[0016] 3. The present invention provides a gene encoding the above-mentioned ribitol dehydrogenase mutant, an expression vector comprising the gene encoding the above-mentioned ribitol dehydrogenase mutant, and a recombinant bacterium comprising the expression vector; the present invention provides a method for preparing and using the above-mentioned ribitol dehydrogenase mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This figure shows the conversion effects of wild-type ribitol dehydrogenase and enzyme mutants on fructose and psicose.

[0018] Figure 2 The reaction process of fructose to sorbitol catalyzed by ribitol dehydrogenase.

[0019] Figure 3 This is a diagram showing the conversion effect of ribitol dehydrogenase mutant (E168D+V172T+F310W) on fructose at different temperatures.

[0020] Figure 4 This is a diagram showing the conversion effect of ribitol dehydrogenase mutant (E168D+V172T+F310W) on fructose at different pH values. DETAILED DESCRIPTION

[0021] The features and exemplary embodiments of various aspects of the present invention will be described in detail below. The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below.

[0022] For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should all be encompassed within the scope of the present application. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can be implemented equally without certain specific details. In other embodiments, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of ​​the present application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0024] In all discussions herein, the standard single-letter codes for amino acids are used. Standard substitution notation is also used, i.e., E168D means that the glutamic acid (E) at position 168 at the N-terminus is replaced by aspartic acid (D). E168D+V172T means that the glutamic acid (E) at position 168 at the N-terminus is replaced by aspartic acid (D), and the valine (V) at position 172 at the N-terminus is replaced by threonine (T).

[0025] The term "wild-type" refers to a gene or gene product that has been isolated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered properties) compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to the wild-type gene or gene product. Methods for introducing or substituting natural, non-naturally occurring amino acids are well known in the art.

[0026] Wild-type ribitol dehydrogenase

[0027] Ribitol dehydrogenases are a class of oxidoreductases that reversibly convert D-ribulose to ribitol. The present invention utilizes publicly available sequence and structural information for ribitol dehydrogenases, performs non-redundant searches in databases such as NCBI, and screens potential ribitol dehydrogenase genes based on protein structural similarity, conserved site analysis, and host-source diversity. These genes are functionally expressed and purified in an Escherichia coli expression system to obtain pure ribitol dehydrogenase. The present invention ultimately selected ribitol dehydrogenase HsRDH, expressed by the ribitol dehydrogenase gene SORD (Gene ID: 6652) from Homo sapiens, as the research object. Ribitol dehydrogenase HsRDH exhibits broad substrate selectivity and can catalyze the conversion of various sugars to their corresponding ribitols. The nucleotide sequence of the ribitol dehydrogenase gene SORD is shown in SEQ ID NO. 2, and the amino acid sequence of the ribitol dehydrogenase HsRDH expressed thereby is shown in SEQ ID NO. 1.

[0028] Ribitol dehydrogenase mutants

[0029] By amplifying the ribitol dehydrogenase gene SORD and using rational design to conduct directed evolution, a ribitol dehydrogenase mutant with high selectivity for fructose as a substrate in a mixed system of fructose and psicose was obtained. The wild-type ribitol dehydrogenase and the ribitol dehydrogenase mutant of the present invention have the ability to catalyze fructose to produce sorbitol. The reaction process is as follows: Figure 2 shown.

[0030] Example

[0031] Example 1 Sequences of ribitol dehydrogenase mutants

[0032] Based on the amino acid sequence SEQ ID NO. 1 of the wild-type ribitol dehydrogenase (WT), seven mutant sequences including E168D, V172T, F310W, E168D+V172T, E168D+F310W, V172T+F310W, and E168D+V172T+F310W were selected.

[0033] Example 2 Construction of Ribitol Dehydrogenase Mutant Expression Vector

[0034] 1. Obtaining ribitol dehydrogenase expression vector and recombinant bacteria

[0035] The wild-type gene for ribitol dehydrogenase from Homo sapiens was synthesized by GenWeiZhi (Suzhou) and constructed on the pET22b vector. The vector was then transformed into the E. coli DH5α strain. The recombinant E. coli DH5α / pET22b-HsRDH was inoculated into a 5 mL test tube filled with culture medium and cultured at 37°C with shaking at 220 rpm for 12 hours. After the incubation period, the cells were centrifuged at 12,000 rpm for 1 minute and harvested. A plasmid was extracted from the E. coli DH5α / pET22b-HsRDH using a high-purity plasmid miniprep kit and used as a template for iterative mutagenesis to construct mutants of the plasmid pET22b-HsRDH.

[0036] 2. Construction of recombinant strains of ribitol dehydrogenase mutants

[0037] The target mutant gene was obtained by whole-plasmid PCR. The primers required were specifically designed using E168D as an example. Other mutants were designed using this principle and single-point iterative mutagenesis was performed.

[0038] E168D upstream primer: gcgctgatt gac ccgctgagc. E168D downstream primer: gctcagcgg gtc The aatcagcgc PCR system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0039] Table 1 PCR reaction system

[0040] Ingredients volume 10×BufferforKOD-Plus- 2.5 μL 2mM dNTP 2.5 μL <![CDATA[25mMMgSO4]]> 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75μL 10 pmol / μL Reverse Primer 0.75μL DNA template <100ng KOD-Plus- 1 μL <![CDATA[ddH2O]]> up to 25 μL

[0041] Table 2 PCR reaction conditions

[0042]

[0043]

[0044] After the PCR amplification was completed, the amplified product was detected by 0.9% agarose gel electrophoresis, and the results showed that the amplified product was a single band with a size of about 6000 bp. The amplified product was purified and recovered using a DNA recovery and purification kit.

[0045] The purified gene fragments were digested with DpnI to remove the template and then recombined using a recombinase. The recombinant product was transformed into E. coli DH5α competent cells and plated on the surface of LB solid medium containing 100 μg / mL ampicillin. The cells were incubated at 37°C for 12 hours, and single colonies were picked and transferred to LB liquid culture. Successful transformants were identified by PCR, and the correctness of the mutation site was verified by sequencing. After verification, a portion of the cells was added with sterile glycerol to a final concentration of 25%, numbered, and stored at -80°C until further use. A portion of the cells was used to extract the plasmid using a plasmid extraction kit, and the recombinant plasmids were stored at -20°C.

[0046] The recombinant expression plasmid pET22b that was successfully sequenced was transferred into E. coli BL21 (DE3) as the expression host to construct the recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-HsRDH.

[0047] Example 3 Cultivation of ribitol dehydrogenase mutant recombinant bacteria and preparation of crude enzyme solution

[0048] The recombinant mutant expression strain E. coli BL21 (DE3) / pET22b-HsRDH was successfully constructed and spread onto a plate containing kanamycin at a final concentration of 100 μg / mL. A single colony was picked and inoculated into 5 mL of LB medium containing resistance and cultured overnight at 37°C at 200 rpm / min. 1% of the inoculum was transferred to 500 mL of LB medium containing resistance and cultured at an OD of 4. 600 When the concentration reaches about 0.6, add IPTG with a final concentration of 0.5 mM and induce at 18°C ​​for about 14 hours.

[0049] After centrifugation, resuspend the cells in buffer and disrupt them by ultrasonication in an ice bath (2-second on-time, 5-second interval, 30-minute on-time). Centrifuge at 12,000 rpm for 20 minutes at 4°C. Collect the supernatant and filter through a 0.22 μm water filter to obtain the crude enzyme solution.

[0050] Example 4: Ribitol dehydrogenase and its mutants catalyze the production of sorbitol from fructose in a mixed system of fructose and psicose

[0051] The crude enzyme solution obtained in Example 3 was used as a catalyst.

[0052] The reaction system is: OD 600=40 crude enzyme solution, 150mM fructose or psicose, 0.5mM NAD + , 500mM sodium formate, commercial formate dehydrogenase at a concentration of 1U / mL, and a 200mM potassium phosphate buffer at pH 7.5. The reaction temperature was controlled at 30°C in a water bath with magnetic stirring for 12 hours. The substrate selectivity and conversion rate of ribitol dehydrogenase and its mutants for fructose and psicose were determined by liquid chromatography. The results are shown in Table 3. Figure 1 .

[0053] Table 3 Conversion efficiency of ribitol dehydrogenase and its mutants to fructose and psicose

[0054]

[0055]

[0056] Example 5: Ribitol dehydrogenase mutant (E168D+V172T+F310W) selectively identifies the optimal temperature for catalyzing fructose to sorbitol in a mixed system of fructose and psicose

[0057] The crude enzyme solution obtained in Example 3 was used as a catalyst.

[0058] The reaction system is: OD 600 =40 crude enzyme solution, 150mM fructose or allulose, 0.5mM NAD + , 500mM sodium formate, 1U / mL commercial formate dehydrogenase, and a 200mM potassium phosphate buffer with a pH of 7.5. The reaction temperature was controlled at 25°C, 30°C, 35°C, 40°C, and 45°C in a water bath with magnetic stirring for 12 hours. The substrate selectivity and conversion rate of ribitol dehydrogenase and its mutants for fructose and psicose were determined by liquid chromatography. The results are shown in Table 1. Figure 3 .

[0059] Experiments have shown that different temperatures have a significant impact on the catalytic activity of HsRDH, with its reaction activity showing a slightly normal trend with temperature changes. The optimal catalytic effect was observed at 30°C, while enzyme activity was relatively low at temperatures below or above this temperature.

[0060] Example 6 Ribitol dehydrogenase mutant (E168D+V172T+F310W) selectively identifies the optimal pH for catalyzing fructose to sorbitol in a mixed system of fructose and psicose

[0061] The crude enzyme solution obtained in Example 3 was used as a catalyst.

[0062] The reaction system is: OD 600= 40 crude enzyme solution, 150mM fructose or psicose, 0.5mM NAD+, 500mM sodium formate, 1U / mL commercial formate dehydrogenase, and the reaction buffer system consisted of 50mM sodium phosphate buffer, pH = 6.5, 200mM potassium phosphate buffer, pH = 7.5, 50mM Tris-HCl buffer, pH = 8, 50mM Tris-HCl buffer, pH = 9, and 50mM boric acid buffer, pH = 10. The reaction temperature was controlled at 30°C in a water bath with magnetic stirring and the reaction was carried out for 12 hours. The substrate selectivity and conversion rate of ribitol dehydrogenase and its mutants for fructose and psicose were detected by liquid chromatography. The results are shown in Figure 5. Figure 4 .

[0063] Experiments have shown that different pH levels significantly affect the catalytic activity of HsRDH, with its activity showing a slightly positive trend with changes in pH. The best catalytic effect was observed at pH 7.5, while enzyme activity decreased under acidic or alkaline conditions.

Claims

1. A ribitol dehydrogenase mutant with improved substrate selectivity, characterized in that: The amino acid sequence is obtained by subjecting the sequence shown in SEQ ID NO. 1 to at least one mutation among E168D, V172T, and F310W.

2. A ribitol dehydrogenase gene, characterized in that: The gene encodes the ribitol dehydrogenase mutant according to claim 1.

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

2.

4. An expression vector, characterized in that The expression vector comprises the nucleotide sequence of claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector is a pET series expression vector.

6. A recombinant bacterium, characterized in that The recombinant bacterium is Escherichia coli containing the expression vector according to claim 4.

7. A method for constructing a recombinant bacterium, characterized in that: The steps include: (1) Construction of an expression vector: ligating the gene described in claim 2 with the enzyme-digested plasmid to obtain a recombinant expression vector; (2) Construction of recombinant bacteria: The constructed recombinant expression vector is transferred into competent Escherichia coli cells, and the recombinant bacteria are cultured and screened to obtain the recombinant bacteria.

8. A catalyst, characterized in that Comprising the ribitol dehydrogenase mutant according to claim 1.

9. Use of the ribitol dehydrogenase mutant according to claim 1 in catalyzing the conversion of fructose into sorbitol.

10. Use according to claim 9, characterized in that The reaction conditions for catalyzing the conversion of fructose into sorbitol include a reaction temperature of 25 to 45° C., a pH of 6.5 to 10, and a reaction time of 6 to 16 hours.

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