Gene for coding glucose dehydrogenase, biological material and application of biological material

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a gene for encoding glucose dehydrogenase, a biological material and application of the gene and the biological material. The invention provides a gene for encoding glucose dehydrogenase. The gene comprises a nucleotide sequence as shown in SEQ ID NO: 1. By expressing the gene, the obtained glucose dehydrogenase can better catalyze NAD < + > or NADP < + > to generate NADPH (Nicotinamide Adenine Dinucleotide Phosphate). According to the invention, escherichia coli Rosetta (DE3) is utilized to express the gene for coding the glucose dehydrogenase, and the activity of the generated active enzyme is 3285.714 U / mg, which is obviously higher than the catalytic activity of the glucose dehydrogenase generated by escherichia coli BL21 (DE3).

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a gene encoding glucose dehydrogenase and a biological material and applications thereof. Background Art

[0002] Coenzyme NAD(P)H plays an important role in various enzyme-catalyzed reactions, especially in redox reactions, which require coenzyme NAD(P)H to participate in the reaction as electron transfer. In the process of product synthesis, a certain amount of coenzyme will be consumed. Therefore, as the reaction proceeds, the intracellular coenzyme content decreases, resulting in a decrease in catalytic efficiency. Since coenzymes are expensive, it is unrealistic to supplement them by exogenous addition. Therefore, regulating the metabolic process of microorganisms through metabolic engineering and increasing the concentration of intracellular NADPH can not only effectively improve production efficiency and reduce costs, but also better ensure the normal progress of the biotransformation process. From a technical and economic perspective, strengthening the coenzyme regeneration cycle is of great significance.

[0003] At present, the main method of metabolic engineering based on the coenzyme NADPH is to enhance the metabolic flux of PPP. In Escherichia coli, glucose has two main metabolic pathways, including the glycolysis pathway (EMP pathway) and the pentose phosphate pathway (PPP pathway). Glucose dehydrogenase (GDH) is widely involved in the glucose metabolic pathway in organisms. + , glucose is oxidized to gluconolactone under the catalysis of GDH, and the coenzyme NAD(P)+ is reduced to NAD(P)H. However, existing glucose dehydrogenases generally have the problem of low activity. Therefore, it is particularly important in this field to optimize the glucose dehydrogenase encoding gene, provide a glucose dehydrogenase with high activity, and then construct a glucose dehydrogenase engineering bacterium. Summary of the invention

[0004] The purpose of the present invention is to provide a gene encoding glucose dehydrogenase and a biomaterial and application thereof, to prepare a high-activity glucose dehydrogenase, to improve the catalytic activity of NAD + or NADP + The efficiency of NADPH generation, especially the increase of NADP + Efficiency of NADPH generation.

[0005] The present invention provides a gene encoding glucose dehydrogenase, wherein the gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0006] The present invention also provides a biological material encoding glucose dehydrogenase, wherein the biological material comprises a recombinant vector and / or a recombinant bacterium;

[0007] The recombinant vector comprises a basic vector and a glucose dehydrogenase encoding gene inserted into the basic vector;

[0008] The recombinant bacteria include a basic strain and the glucose dehydrogenase encoding gene or the recombinant vector introduced into the basic strain;

[0009] The glucose dehydrogenase encoding gene is the gene described in the above technical solution.

[0010] Preferably, the basic vector comprises pBAD-HISA;

[0011] The basic strain includes Escherichia coli Rosetta (DE3).

[0012] The present invention also provides a method for expressing glucose dehydrogenase, which induces expression of a strain containing the gene described in the above technical solution to obtain NAD kinase.

[0013] Preferably, the induced expression comprises inducing expression using arabinose.

[0014] Preferably, the final concentration of arabinose is 0.1-0.5 wt.%.

[0015] Preferably, after the induced expression, the method further comprises: purifying the induced expression product.

[0016] The present invention also provides the gene or the biomaterial or the expression method in the above technical solution in catalyzing NAD + or NADP + Application in generating NADPH;

[0017] The NAD + or NADP + The substrate for the reaction to generate NADPH is glucose.

[0018] The present invention also provides a catalytic NAD + or NADP + The method of generating NADPH is + or NADP + Glucose dehydrogenase expressed by recombinant bacteria is added to the system for generating NADPH for reaction;

[0019] The recombinant bacteria include a basic strain and a glucose dehydrogenase encoding gene or the recombinant vector introduced into the basic strain; the glucose dehydrogenase encoding gene is the gene described in the above technical solution;

[0020] The basic strain includes Escherichia coli Rosetta (DE3).

[0021] Preferably, the NAD + or NADP +The system for generating NADPH includes 50-300 μM NAD + / NADP + , 200-1000 mM glucose, 0.01-1 mg / mL glucose dehydrogenase and the balance PBS buffer;

[0022] The reaction temperature is 20-37° C. and the reaction time is 3-10 min.

[0023] Beneficial effects:

[0024] The present invention provides a gene encoding glucose dehydrogenase, wherein the gene comprises a nucleotide sequence as shown in SEQ ID NO: 1. By expressing the gene, the glucose dehydrogenase obtained by the present invention can better catalyze NAD + or NADP + Generate NADPH. The results of the embodiment show that the activity of the active enzyme produced by expressing the gene encoding glucose dehydrogenase in Escherichia coli Rosetta (DE3) is 3285.714 U / mg. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 This is a gel image of glucose dehydrogenase (GDH);

[0027] Figure 2 The results of the GDH activity test produced by E. coli BL21 (DE3);

[0028] Figure 3 The results of GDH activity detection produced by Escherichia coli Rosetta (DE3) are shown. DETAILED DESCRIPTION

[0029] The present invention provides a gene encoding glucose dehydrogenase, wherein the gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0030] The present invention also provides a biological material encoding glucose dehydrogenase, the biological material comprising a recombinant vector and / or a recombinant bacterium; the recombinant vector comprising a basic vector and a glucose dehydrogenase encoding gene inserted into the basic vector; the recombinant bacterium comprising a basic strain and the glucose dehydrogenase encoding gene or the recombinant vector introduced into the basic strain; the glucose dehydrogenase encoding gene is the gene described in the above technical solution.

[0031] As an embodiment, the basic vector includes pBAD-HISA. As an embodiment, the gene encoding glucose dehydrogenase is inserted between the Nco I and Xho I restriction sites of the pBAD-HISA. The present invention utilizes pBAD-HISA to accelerate the expression of glucose dehydrogenase compared to other basic vectors.

[0032] As an embodiment, the basic strain includes Escherichia coli Rosetta (DE3). The present invention utilizes Escherichia coli Rosetta (DE3), and the glucose dehydrogenase expressed by the strain has higher activity than other strains (such as Escherichia coli BL21 (DE3)).

[0033] The present invention also provides a method for expressing glucose dehydrogenase, which induces expression of a strain containing the gene described in the above technical solution to obtain NAD kinase.

[0034] The present invention induces expression of a strain containing the gene encoding glucose dehydrogenase described in the above technical solution to obtain an induced expression product; the induced expression product contains glucose dehydrogenase.

[0035] As an embodiment, the induced expression includes inducing expression using arabinose. As an embodiment, the final concentration of arabinose is 0.1-0.5 wt. %; as another embodiment, the final concentration of arabinose is 0.2 wt. %.

[0036] As an embodiment, after obtaining the induced expression product, the present invention purifies the induced expression product. As an embodiment, the purification of the present invention uses a Ni column. The present invention has no strict requirements on the specific steps of the purification, and conventional methods in the art can be used.

[0037] The present invention constructs a gene library of glucose dehydrogenase (GDH) and screens a gene encoding glucose dehydrogenase (GDH gene), the nucleotide sequence of which is shown in SEQ ID NO: 1. By expressing the gene, the obtained glucose dehydrogenase has better catalytic performance of NAD + or NADP + NADPH-generating activity.

[0038] In view of this, the gene or the biomaterial or the expression method described in the above technical solution catalyzes NAD + or NADP + Application in generating NADPH; the NAD + or NADP + The substrate for the reaction to generate NADPH is glucose.

[0039] The present invention also provides a catalytic NAD + or NADP + The method of generating NADPH is + or NADP + Glucose dehydrogenase expressed by recombinant bacteria is added to the system for generating NADPH for reaction;

[0040] The recombinant bacteria include a basic strain and a glucose dehydrogenase encoding gene or the recombinant vector introduced into the basic strain; the glucose dehydrogenase encoding gene is the gene described in the above technical solution;

[0041] The basic strain includes Escherichia coli Rosetta (DE3).

[0042] As an embodiment, the NAD + or NADP + The system for generating NADPH includes 50-300 μM NAD + / NADP + , 200-1000 mM glucose, 0.01-1 mg / mL glucose dehydrogenase, and the remainder PBS buffer; as another embodiment, the NAD + or NADP + The system for generating NADPH includes 200 μM NAD + / NADP + , 500 mM glucose, 0.034 mg / mL glucose dehydrogenase, and the balance PBS buffer.

[0043] As an embodiment, the reaction temperature is 20-37° C.; as another embodiment, the reaction temperature is 25-28° C. As an embodiment, the reaction time is 3-10 min; as another embodiment, the reaction time is 5 min.

[0044] To further illustrate the present invention, a gene encoding glucose dehydrogenase and a biological material and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The inventor team constructed a gene library of glucose dehydrogenase (GDH), and screened out a glucose dehydrogenase encoding gene (GDH gene). The nucleotide sequence is: 5'-ATGCCGGCGCCGTATAAAGATCGTTTTGCGGGCAAAAAAGTGCTGGTGA CCGGCGCGAGCCAGGGCATTGGCGAAGCGACCGCGCTGCGTTTTGCGGAAGAAGGCGCGCAGGTGGCGCTGAACGGCCGTAAAGAAGATAAACTGATTGCGGTGCGTGAAAAACTGCCGAAAGTGAGCGGCGGCGAACATCCGATTGCGACCGGCGATATTAGCAAAGAAGATGATGTGAAACGTCTGGTGGCGGAAAGCATTAAAGCGATGGGCGGCCTGGATGTGCTGGTGTGCAACGCGGGCTATCAGATTCCGAGCCCGAGCGAAGATATTAAACTGGAAGATTTTGAAGGCGTGATGGCGGTGAACGTGACCGGCGTGATGCTGCCGTGCCGTGAAGTGATTCGTTATTGGCTGGAAAACGGCATTAAAGGCACCATTATTGTGAACAGCAGCGTGCATCAGATTATTCCGAAACCGCATTATCTGGGCTATAGCGCGAGCAAAGGCGCGGTGGGCAACATTGTGCGTACCCTGGCGCTGGAATATGCGACCCGTGGCATTCGTGTGAACGCGGTGGCGCCGGGCGCGATTGTGACCCCGATTAACATGAGCTGGATTGATGATCCGGAACAGTATAAAGCGGTGAGCAGCCATATTCCGATGAAACGTCCGGGCGAAAGCCGTGAAATTGCGGATGCGATTACCTTTCTGGCGGCGGAAGATAGCACCTATATTACCGGCCAGACCCTGTATGTGGATGGCGGCCTGACCCTGTATGGCGATTTTGAAAACAACTGGAGCAGCTGA-3'

[0047] (SEQ ID NO: 1), and the amino acid sequence of the glucose dehydrogenase encoded by it is: MPAPYKDRFAGKKVLVTGASQGIGEATALRFAEEGAQVALNGRKEDKLIAVREKLPKVSGGEHPIATGDISKEDDVKRLVAESIKAMGGLDVLVCNAGYQIPSPSEDIKLEDFEGVMAVNVTGVMLPCREVIRYWLENGIKGTIIVNSSVHQIIPKPHYLGYSASKGAVGNIVRTLALEYATRGIRVNAVAPGAIVTPINMSWIDDPEQYKAVSSHIPMKRPGESREIADAITFLAAEDSTYITGQTLYVDGGLTLYGDFENNWSS (SEQ ID NO: 2).

[0048] Example 2

[0049] 1. Implementation Materials

[0050] (1) Expression vector: pBAD-HISA(Amp), arabinose induction;

[0051] (2) Expression host: Escherichia coli BL21 (DE3); Escherichia coli Rosetta (DE3);

[0052] (3) PBS buffer (pH 7.2-7.4): 2 mM disodium hydrogen phosphate, 0.4 mM potassium dihydrogen phosphate, 0.54 mM potassium chloride, 27.4 mM sodium chloride and 250 mM imidazole buffer (containing PBS buffer, pH 8.0).

[0053] 2. Construction of recombinant expression vector

[0054] The coding gene obtained in Example 1 was inserted between the Nco I and Xho I restriction sites of the expression vector, and positive clones were screened to obtain a recombinant expression vector.

[0055] 3. Construction of recombinant expression bacteria

[0056] (1) The recombinant expression vector in step 2 was transformed into Escherichia coli BL21 (DE3) (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the positive cell strains were screened to obtain the recombinant expression bacteria 1;

[0057] (2) The recombinant expression vector of step 2 was transformed into Escherichia coli Rosetta (DE3) (purchased from Bio-Time), and the positive cell lines were screened to obtain the recombinant expression bacteria 2.

[0058] 4. Inducible Expression

[0059] In 500 mL of LB medium (containing the corresponding resistance), inoculate the seed solution of recombinant expression bacteria 1 and 2 in step 3 at a 1% inoculum volume, and culture on a shaking incubator (37°C, 220 rpm) for 3-5 h until the OD 600 About 0.6-0.8; arabinose was added to a final concentration of 0.2wt.%, and induced at 30°C overnight.

[0060] 5. Purification

[0061] The fermentation broth obtained in step 4 was centrifuged at 6000 rpm for 30 min, and the supernatant was discarded to retain the bacterial cells; PBS buffer (pH 7.2-7.4) was added to the bacterial cells, and the bacterial cells were broken by a high-pressure homogenizer; the broken liquid was centrifuged at 15000 rpm for 30 min to remove bacterial fragments; the broken liquid after centrifugation was further filtered through a 0.22 μm filter membrane to further remove large particle impurities, and two kinds of filtered crude enzyme solutions were obtained; Ni column purification was performed according to the following steps:

[0062] (1) Equilibrate the column: wash with PBS buffer (pH 7.2-7.4);

[0063] (2) Loading: Load the filtered crude enzyme solution;

[0064] (3) Equilibrate the column: wash with PBS buffer containing 10 mM imidazole;

[0065] (4) Obtaining the target protein: gradient elution was performed using PBS buffer with different concentrations of imidazole. The elution procedure was as follows: solution A was PBS, and solution B was PBS buffer containing 500 mM imidazole; when the total volume was 135 mL, solution B was 100%, and the elution rate was 3 mL / min.

[0066] (5) Column storage: After obtaining the protein, the Ni column was rinsed with ultrapure water and stored in a 20% (v / v) ethanol solution.

[0067] 6. Concentration

[0068] During the elution process, a peak graph appeared at about 12 minutes, and the corresponding eluate was collected for SDS detection. The results showed that the size of glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 1 and 2 was about 28KDa; among them, the gel graph of glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 2 is as follows Figure 1 Select a 10KDa ultrafiltration tube to remove the imidazole eluate by centrifugation and concentrate the protein, and detect the protein concentration using a Bradford kit.

[0069] Example 3

[0070] Activity detection

[0071] 1. Glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 1 in Example 2 was used as the test sample for activity detection, wherein the activity detection system was as follows: 200 μM NAD + , 500 mM glucose (Glu), 0.034 mg / mL test sample, PBS buffer (pH 7.2) is added to 40 mL, and the reaction is carried out at 25°C.

[0072] 2. The glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 1 in Example 2 was used as the test sample for activity detection, wherein the activity detection system is as follows: 200 μM NADP + , 500 mM glucose (Glu), 0.034 mg / mL test sample, PBS buffer (pH 7.2) is added to 40 mL, and the reaction is carried out at 25°C.

[0073] 3. During the reaction of steps 1 to 2, samples were taken at a fixed point (after 5 minutes of reaction) to detect enzyme activity; the liquid volume of the cuvette was 1000 μL, and the test wavelength was 340 nm. The test results of glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 1 are as follows: Figure 2 As shown; the detection results of glucose dehydrogenase (GDH) obtained by recombinant expression bacteria 2 are as follows Figure 3 shown.

[0074] according to Figure 2 It can be seen that glucose dehydrogenase expressed by recombinant expression bacteria 1 (Escherichia coli BL21 (DE3)) is involved in catalyzing NAD + or NADP + The activity of the reaction to generate NADPH is weak, with an absorbance of less than 0.05. The glucose dehydrogenase expressed by Escherichia coli BL21 (DE3) has poor activity; the glucose dehydrogenase expressed by recombinant expression bacteria 2 (Escherichia coli Rosetta (DE3)) participates in the catalysis of NADP + The reaction of generating NADPH is very active, with an absorbance of 1.2. The glucose dehydrogenase expressed by E. coli Rosetta (DE3) has good activity. The amount of enzyme required to generate 1 μM NADPH within 1 minute is defined as U. The activity of active glucose dehydrogenase produced by Rosetta (DE3) is 3285.714 U / mg, and that of BL21 (DE3) is 0.237 U / mg, which is significantly different from each other.

[0075] From the above content, it can be seen that the activity of glucose dehydrogenase produced by Escherichia coli Rosetta (DE3) is stronger than that produced by Escherichia coli BL21 (DE3).

[0076] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene encoding glucose dehydrogenase, characterized in that The gene comprises the nucleotide sequence shown in SEQ ID NO:

1.

2. A biological material encoding glucose dehydrogenase, characterized in that The biological material includes a recombinant vector and / or a recombinant bacterium; The recombinant vector comprises a basic vector and a glucose dehydrogenase encoding gene inserted into the basic vector; The recombinant bacteria include a basic strain and a glucose dehydrogenase encoding gene introduced into the basic strain or the recombinant vector; The glucose dehydrogenase encoding gene is the gene according to claim 1.

3. The biomaterial according to claim 2, characterized in that The basic vector includes pBAD-HISA; The basic strain includes Escherichia coli Rosetta (DE3).

4. A method for expressing glucose dehydrogenase, characterized in that: The strain containing the gene according to claim 1 is induced to express to obtain NAD kinase.

5. The expression method according to claim 4, characterized in that: The induced expression includes inducing expression using arabinose.

6. The expression method according to claim 5, characterized in that: The final concentration of the arabinose is 0.1-0.5 wt.%.

7. The expression method according to any one of claims 4 to 6, characterized in that: After the induced expression, the method further comprises: purifying the product of the induced expression.

8. The gene according to claim 1, the biological material according to claim 2 or 3, or the expression method according to any one of claims 4 to 7 in catalyzing NAD + or NADP + Application in generating NADPH; The NAD + or NADP + The substrate for the reaction to generate NADPH is glucose.

9. A catalytic NAD + or NADP + A method for generating NADPH, characterized in that At NAD + or NADP + Glucose dehydrogenase expressed by recombinant bacteria is added to the system for generating NADPH for reaction; The recombinant bacteria include a basic strain and a glucose dehydrogenase encoding gene or the recombinant vector introduced into the basic strain; the glucose dehydrogenase encoding gene is the gene according to claim 1; The basic strain includes Escherichia coli Rosetta (DE3).

10. The method according to claim 9, characterized in that The NAD + or NADP + The system for generating NADPH includes 50-300 μM NAD + / NADP + , 200-1000 mM glucose, 0.01-1 mg / mL glucose dehydrogenase and the balance PBS buffer; The reaction temperature is 20-37° C. and the reaction time is 3-10 min.

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