Recombinant glucose dehydrogenase as well as preparation method therefor and use thereof

Abstract

A recombinant glucose dehydrogenase as well as a preparation method therefor and the use thereof. Specifically, the present invention comprises using genetically engineered Escherichia coli to express the recombinant glucose dehydrogenase. Soluble expression of the recombinant glucose dehydrogenase in the Escherichia coli system enables an expressed target protein to have high yield and high catalytic activity. Therefore, the present invention has the advantages of short production period, easy purification of expression products, low production cost and the like, and can realize industrial production of the recombinant glucose dehydrogenase.

Description

Recombinant glucose dehydrogenase and its preparation method and application

[0001] Cross-reference to related applications

[0002] This patent application claims priority to the Chinese patent application filed on December 13, 2023, with application number 202311713543.3 and invention name “Recombinant glucose dehydrogenase, preparation method and application thereof”. The full text of the above application is incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant glucose dehydrogenase and a preparation method thereof. Background Art

[0004] Glucose dehydrogenase (GDH) is widely present in prokaryotes and eukaryotes. It can be divided into three types based on its different coenzyme binding types: (1) pyrroquinoline quinone-dependent (PQQ-GDH; EC 1.1.5.2); (2) flavin adenine dinucleotide-dependent (FAD-dependent, FAD-GDH; EC 1.1.99.10); and (3) nicotinamide adenine dinucleotide-dependent (NAD(P)+-dependent, NAD(P)+-GDH; EC 1.1.1.47). GDH is a member of the short-chain alcohol dehydrogenase family and consists of four identical subunits (approximately 30 kd). In the presence of the coenzyme NAD(P)+, it can catalyze the conversion of β-D-glucose to D-glucono-δ-lactone. This enzyme is a key enzyme in the sporulation stage of Bacillus. In recent years, it has been widely studied and applied in many fields. It can be used in biofuel cells, bioreactors for clinical detection of blood glucose levels, and coenzyme regeneration systems in large-scale chiral synthesis.

[0005] GDH catalyzes the oxidation of glucose to produce gluconic acid, while simultaneously combining the generated hydrogen ions with the electron acceptor NAD+ or NADP+ to generate the reduced coenzyme NADH or NADPH, thereby achieving cyclic regeneration of the reduced coenzyme. Due to its low substrate cost, it has become the most widely used coenzyme regeneration technology. Blood glucose level is the main and routine test indicator for the clinical diagnosis of diabetes. Glucose dehydrogenase has been found to have the potential to replace glucose oxidase. Because it does not use oxygen as an electron acceptor and is not restricted by dissolved oxygen, the test results have smaller errors. In 2008, Okuda-Shimazaki constructed a biofuel cell using heat-stable glucose dehydrogenase. The battery showed that the output power depends on the glucose concentration. Currently, GDH is mainly extracted from animal liver, and the source is limited.

[0006] Therefore, those skilled in the art are committed to transforming and recombinantly expressing glucose dehydrogenase in order to prepare low-cost glucose dehydrogenase that is suitable for industrial production and can maintain high reaction activity.

[0007] Summary of the Invention

[0008] The object of the present invention is to provide a recombinant glucose dehydrogenase and a preparation method thereof.

[0009] In the first aspect of the present invention, a recombinant glucose dehydrogenase is provided, the amino acid sequence of the recombinant glucose dehydrogenase is shown in SEQ ID NO.1, or the amino acid sequence of the recombinant glucose dehydrogenase has at least 80% homology compared with SEQ ID NO.1; more preferably, it has at least 90% homology; most preferably, it has at least 95% homology; such as at least 96%, 97%, 98%, or 99% homology.

[0010] The second aspect of the present invention provides a polynucleotide molecule encoding the recombinant glucose dehydrogenase described in the first aspect of the present invention.

[0011] In another preferred embodiment, the polynucleotide molecule sequence is shown as SEQ ID NO.2.

[0012] The third aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0013] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the first aspect of the present invention or the chromosome is integrated with the nucleic acid molecule described in the second aspect of the present invention.

[0014] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0015] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0016] The fifth aspect of the present invention provides a method for preparing the recombinant glucose dehydrogenase according to the first aspect of the present invention, comprising the steps of:

[0017] (i) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to express the recombinant glucose dehydrogenase; and

[0018] (ii) isolating the recombinant glucose dehydrogenase.

[0019] In another preferred embodiment, the temperature for culturing the host cells in step (i) is 20°C-40°C; preferably 25°C-37°C, such as 37°C.

[0020] In another preferred embodiment, the host cell in step (i) is an Escherichia coli cell.

[0021] The sixth aspect of the present invention provides a kit, which comprises the recombinant glucose dehydrogenase described in the first aspect of the present invention.

[0022] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the electrophoresis results of recombinant glucose dehydrogenase expression;

[0024] Figure 2 is a standard curve diagram for enzyme activity detection. DETAILED DESCRIPTION

[0025] Through extensive and in-depth research, the inventors have achieved soluble expression of recombinant glucose dehydrogenase in genetically engineered Escherichia coli, with high protein yield and high catalytic activity. This method offers advantages such as a short production cycle, easy purification of the expressed product, and low cost, enabling industrialized production of recombinant glucose dehydrogenase. Based on this, the present invention was completed.

[0026] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0028] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0029] In a preferred embodiment of the present invention, the amino acid sequence of the recombinant glucose dehydrogenase according to the present invention (SEQ ID NO.1) is as follows:

[0030] In a preferred embodiment of the present invention, the gene sequence of the recombinant glucose dehydrogenase is as follows (optimized by synonymous codon preference of Escherichia coli, SEQ ID NO. 2):

[0031] Those skilled in the art can obtain the recombinase gene sequence of the present invention using conventional methods, such as complete artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers to produce a large amount of single-stranded DNA (ssDNA) after PCR amplification. These primers are referred to as the non-restricting primer and the restricting primer, respectively, and their ratio is generally 50-100:1. In the first 10-15 cycles of the PCR reaction, the amplified product is primarily double-stranded DNA. However, once the restricting primer (low-concentration primer) is consumed, PCR guided by the non-restricting primer (high-concentration primer) produces a large amount of single-stranded DNA. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0032] The mutant enzyme of the present invention can be expressed or produced by conventional recombinant DNA technology, comprising the steps of:

[0033] (1) transforming or transducing a suitable host cell with a polynucleotide encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0034] (2) culturing the host cells in a suitable culture medium;

[0035] (3) Isolate and purify the target protein from the culture medium or cells to obtain the target enzyme.

[0036] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the enzyme of the present invention and appropriate transcriptional / translational control signals, preferably the commercially available vector pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcriptional terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

[0037] The recombinant vector comprises, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If necessary, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polyadenylation signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selectable marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0038] Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0039] Those skilled in the art can construct vectors containing the promoter of the present invention and / or target gene sequence using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0040] The expression vectors of the present invention can be used to transform appropriate host cells to cause the host to transcribe the target RNA or express the target protein. The host cells can be prokaryotes such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, or Agrobacterium; lower eukaryotic cells such as yeast cells; or higher eukaryotic cells such as plant cells. Those skilled in the art will appreciate how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), CaCl2 treatment or electroporation can be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, the leaf disc method, the embryo transformation method, and the flower bud immersion method. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0041] The term "operably linked" means that the target gene to be transcribed and expressed is linked to its control sequence in a conventional manner in the art so as to be expressed.

[0042] Cultivation of engineered bacteria and fermentation production of target protein

[0043] After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention. Depending on the host cell, the culture medium used can be selected from various conventional culture media, and the cells are cultured under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0044] In the present invention, conventional fermentation conditions can be used. Representative conditions include (but are not limited to):

[0045] (a) Regarding temperature, the fermentation and induction temperature of the enzyme is maintained at 25-37°C;

[0046] (b) With regard to the pH value during the induction period, the pH during the induction period is controlled at 3-9;

[0047] (c) Dissolved oxygen (DO) is controlled at 10-90%. This can be maintained by introducing an oxygen / air mixture.

[0048] (d) As for feed, feed types should preferably include carbon sources such as glycerol, methanol, and glucose, which can be fed individually or in combination;

[0049] (e) With respect to the IPTG concentration during the induction period, conventional induction concentrations can be used in the present invention, and the IPTG concentration is usually controlled at 0.1-1.5 mM;

[0050] (f) The induction time is not particularly limited, but is usually 2 to 20 hours, preferably 5 to 15 hours.

[0051] The target protein of the present invention is present intracellularly in Escherichia coli cells. The host cells are collected by centrifugation and then disrupted by high pressure, mechanical force, enzymatic cell lysis, or other cell disruption methods to release the recombinant protein. High pressure is preferred. The host cell lysate can be initially purified by methods such as flocculation, salting out, and ultrafiltration, followed by purification by chromatography, ultrafiltration, or directly by chromatography.

[0052] Chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Commonly used chromatography methods include:

[0053] 1. Anion exchange chromatography:

[0054] Anion exchange chromatography media include (but are not limited to) Q-Sepharose and DEAE-Sepharose. If the fermentation sample has a high salt concentration that affects binding to the ion exchange media, the salt concentration should be reduced before ion exchange chromatography. The sample can be equilibrated with a buffer exchanger using dilution, ultrafiltration, dialysis, gel filtration chromatography, or other methods until the equilibration buffer system matches that of the corresponding ion exchange column. The sample can then be loaded and eluted using a salt concentration or pH gradient.

[0055] 2. Hydrophobic chromatography:

[0056] Hydrophobic chromatography media include (but are not limited to) phenyl-sepharose, butyl-sepharose, and octyl-sepharose. The sample is loaded with a solution containing a salt concentration increased by adding NaCl, (NH₄)₂SO₄, or other methods. Elution is then performed by decreasing the salt concentration. Hydrophobic chromatography removes contaminating proteins with significantly different hydrophobicities.

[0057] 3. Gel Filtration Chromatography

[0058] Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, Sephadex, etc. Gel filtration chromatography can be used to replace the buffer system or further purify the product.

[0059] 4. Affinity chromatography

[0060] Affinity chromatography media include (but are not limited to): HiTrap TM HeparinHPColumns.

[0061] 5. Membrane filtration

[0062] Ultrafiltration media include organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Purification and concentration can be achieved through membrane filtration.

[0063] The main advantages of the present invention are:

[0064] (1) The recombinant glucose dehydrogenase of the present invention can be expressed in large quantities in an E. coli expression system, and thus can be easily purified after fermentation, with a high yield, and the purified protein has high activity.

[0065] (2) The present invention uses genetically engineered Escherichia coli to express recombinant glucose dehydrogenase, providing a method for industrial production of recombinant glucose dehydrogenase. This method has the advantages of simple purification steps, high enzyme activity, high protein expression, and low production cost.

[0066] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, are generally performed according to conventional conditions such as those described in "Molecular Cloning Laboratory Manual" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0067] Example 1 Expression and purification of recombinant protein

[0068] The wild-type glucose dehydrogenase gene sequences from Bacillus Root147 (Sequence ID: KRD84782.1), Acetobacter senegalensis, Escherichia coli, Bacillus subtilis, and Pseudomonas sp. were optimized for synonymous codon usage in E. coli and ligated into the pET-28a(+) vector. The gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and expressed in E. coli BL21(DE3).

[0069] The results showed that glucose dehydrogenases from Pseudomonas aeruginosa, Acetobacter senegal, and Bacillus Root147 were mainly expressed in inclusion bodies in E. coli expression strains, while glucose dehydrogenases from E. coli and Bacillus subtilis could be expressed in the supernatant of E. coli expression strains; qualitative activity test of bacterial lysate showed that only crude glucose dehydrogenases from E. coli and Bacillus Root147 were active, so glucose dehydrogenases from E. coli and Bacillus Root147 were selected for subsequent experiments.

[0070] Based on the wild-type Bacillus Root147-derived glucose dehydrogenase protein sequence (KRD84782.1), its structure was optimized using computer molecular simulation to avoid inclusion body expression in Escherichia coli. The codon optimization of the recombinant glucose dehydrogenase protein after amino acid optimization was then performed to improve the protein expression and enzyme activity of the recombinant glucose dehydrogenase.

[0071] The optimized glucose dehydrogenase protein sequence is as follows (SEQ ID NO.1):

[0072] 1) Escherichia coli synonymous codon preference optimized base sequence

[0073] Gene sequence 1 of the codon-optimized recombinant glucose dehydrogenase (SEQ ID NO. 2):

[0074] Gene sequence 2 of the codon-optimized recombinant glucose dehydrogenase (SEQ ID NO.3):

[0075] Gene sequence 3 of the codon-optimized recombinant glucose dehydrogenase (SEQ ID NO.4):

[0076] 2) In accordance with the experimental design requirements of the present invention, the sequence after optimization of the synonymous codon preference of Escherichia coli was connected to the vector pET-28a(+), which was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0077] 3) Introducing the recombinant plasmid into the host Escherichia coli

[0078] Take 1 μL of the expression plasmid and add it to 30 μL of competent E. coli BL21 (DE3) on ice. Place the cells on ice for 30 minutes, then in a 42°C water bath for 45 seconds. Immediately place the cells on ice for 2 minutes. Add 400 μL of antibiotic-free SOC medium and incubate at 37°C with shaking at 230 rpm for 45 minutes. Spread 100 μL of the bacterial solution evenly onto an LB plate containing 100 μg / mL kanamycin and incubate overnight at 37°C.

[0079] 4) Target gene expression process

[0080] Single clones from step 2) were selected and aseptically inoculated into LB medium containing 100 μg / mL kanamycin (Kanamycin) in duplicate. Cultured at 37°C with shaking at 220 rpm until the OD600 reached 0.6-0.8. Induced with IPTG, the cells were incubated at 37°C with shaking overnight. Samples were then sonicated and analyzed by SDS-PAGE.

[0081] Figure 1 shows the expression test results of different codons for recombinant glucose dehydrogenase from Bacillus. Lane 1 is a molecular weight marker, lane 2 is wild-type glucose dehydrogenase, lane 3 is codon-optimized gene sequence 1, lane 4 is codon-optimized gene sequence 2, and lane 5 is codon-optimized gene sequence 3.

[0082] 4) Ni column purification

[0083] Dialysis Buffer: 10x PBS diluted to 1x, pH 7.4

[0084] Weigh approximately 4 g of cells and add 20 ml of Lysis Buffer to the suspension. Disperse the cells using a dispersant on ice. Ultrasonicate the cells using a Φ10 probe, 10% power, 5.5 seconds on, 9.9 seconds off, and 30 minutes of sonication. Centrifuge at 20,000 rpm at 4°C for 30 minutes, collect the supernatant, and filter through a 0.22 μm membrane. Purify with 1 ml of Ni-NTA at a flow rate of 0.5 ml / min. After loading, rinse the UV and conductivity levels to baseline with 20 ml of Lysis Buffer. Elution procedure: Step 1: 0% B, 10 CV, 2 ml / min; Step 2: 0-60% B, 20 CV, 2 ml / min; Step 3: 100% B, 10 CV, 0 ml / min. Collect the sample and perform electrophoresis.

[0085] Electrophoresis results indicate that the purified target protein is highly pure. Based on the SDS results, select the purified sample collection solution, place it in a 10 kDa dialysis bag, and dialyze it overnight in 1300 ml of dialysate. The next morning, collect the dialyzed sample, mix it, and prepare the target protein sample. Store it in a -80°C freezer. Determine the concentration using the BCA assay.

[0086] The concentration of the codon-optimized gene sequence 1 expression strain sample was measured to be 3.23 mg / ml, the yield was 62.3 mg, and the yield was 15.575 mg / g bacteria.

[0087] The concentration of the codon-optimized gene sequence 2 expression strain sample was measured to be 1.318 mg / ml, the yield was 34.27 mg, and the yield was 8.567 mg / g bacteria.

[0088] The concentration of the codon-optimized gene sequence 3 expression strain sample was measured to be 1.625 mg / ml, the yield was 40.26 mg, and the yield was 10.065 mg / g bacteria.

[0089] Example 2 Determination of glucose dehydrogenase activity

[0090] 1) Solution preparation

[0091] ① Reagent 2: Dissolve in 15 ml of reagent 1 and store at 4°C.

[0092] ② Reagent 3: Add 5 ml of reagent 1 to dissolve and store at -20℃.

[0093] ③ Preparation of working solution: Mix Reagent 1, Reagent 2, and Reagent 3 in a volume ratio of 4:3:2 and use immediately. Reagents 1, 2, and 3 are provided by the Glucose Dehydrogenase (GCDH) Assay Kit (Solarbio, BC2690).

[0094] 2) Experimental steps

[0095] ① Preparation of positive enzyme: Dissolve 10 mg (277 U / mg) of positive enzyme in 100 μL PBS pH 7.4 buffer to prepare 28 U / μL enzyme solution, and then dilute it step by step according to the gradient. The diluent is PBS pH 7.4 buffer.

[0096] ② Preheat the microplate reader for 30 minutes, set the temperature to 37°C, and preheat the working solution at 37°C for 10 minutes.

[0097] ③ Microplate reader program setting: add 100 μL of reaction solution, detect the absorbance value A1 at 340 nm, then add 1 μL of enzyme solution of each concentration, incubate at 37°C for 1 min 30 s, and detect the absorbance value A2 at 340 nm.

[0098] ④Calculate the OD difference between the sample and the blank A2-A1

[0099] The results of the glucose dehydrogenase standard curve are shown in Figure 2.

[0100] The results of enzyme activity detection of each expression strain are as follows:

[0101] The results showed that the glucose dehydrogenase expressed by codon-optimized gene sequence 1 had the highest specific activity, the glucose dehydrogenase expressed by codon-optimized gene sequence 2 had a higher specific activity, and the glucose dehydrogenase expressed by codon-optimized gene sequence 3 had a significantly lower specific activity than other codons.

[0102] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A recombinant glucose dehydrogenase, characterized in that: The amino acid sequence of the recombinant glucose dehydrogenase is shown in SEQ ID NO.1, or the amino acid sequence of the recombinant glucose dehydrogenase has at least 80% homology with SEQ ID NO.

1.

2. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the recombinant glucose dehydrogenase according to claim 1.

3. The polynucleotide molecule according to claim 2, characterized in that The sequence of the polynucleotide molecule is shown as SEQ ID NO.

2.

4. A carrier, characterized in that The vector contains the polynucleotide molecule according to claim 2.

5. A host cell, characterized in that The host cell contains the vector of claim 4 or the polynucleotide molecule of claim 2 is integrated into the chromosome.

6. The host cell according to claim 5, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

7. A method for preparing the recombinant glucose dehydrogenase according to claim 1, characterized in that: Includes steps: (i) culturing the host cell according to claim 5 under suitable conditions to express the recombinant glucose dehydrogenase; and (ii) isolating the recombinant glucose dehydrogenase.

8. The method according to claim 7, characterized in that The temperature of culturing the host cells in step (i) is 20°C-40°C 9. The method according to claim 7, characterized in that The host cell in step (i) is an Escherichia coli cell.

10. A kit, characterized in that: The kit comprises the recombinant glucose dehydrogenase according to claim 1.

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