A novel NAD-dependent glucose-6-phosphate dehydrogenase and its applications

Abstract

This invention relates to the field of biotechnology, providing a novel NAD-dependent glucose-6-phosphate dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.3. The invention also provides the encoding gene of the above-mentioned gluconate-6-phosphate dehydrogenase, a recombinant expression vector including this gene, and a recombinant engineered bacterium. Furthermore, the invention provides a method for preparing the above-mentioned gluconate-6-phosphate dehydrogenase and its application in starch hydrogen production. This invention obtains a novel NAD-dependent NAD-dependent gluconate-6-phosphate dehydrogenase by mutating wild-type gluconate-6-phosphate dehydrogenase. + A mutant with better enzyme activity; this mutant is an NAD-dependent glucose-6-phosphate dehydrogenase, which can be used for hydrogen production from starch, effectively reducing the cost of hydrogen production and facilitating industrial production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a novel NAD-dependent glucose-6-phosphate dehydrogenase and its application in starch-to-hydrogen production. Background Technology

[0002] Traditional fossil fuels (including oil, natural gas, and coal) are currently the world's primary energy sources. However, due to their non-renewable and rapidly depleted nature, as well as the severe environmental impacts caused by pollutants such as greenhouse gases, ozone-depleting substances, and acid oxides produced during combustion, these problems constitute a serious global challenge. As a carbon-free clean fuel, hydrogen is considered an ideal energy carrier for future sustainable development due to its high efficiency and environmentally friendly characteristics. Unlike traditional fossil fuels, hydrogen only produces water during combustion or electrochemical reactions, without generating greenhouse gases, ozone-depleting substances, or other harmful pollutants, thus possessing significant environmental advantages.

[0003] However, global hydrogen production still mainly relies on traditional fossil fuel-based hydrogen production technologies. These processes not only have low energy conversion efficiency but also involve large amounts of carbon dioxide emissions, and are therefore generally considered to be inconsistent with the requirements of green and sustainable development.

[0004] Biomass-based hydrogen production is the process of producing hydrogen from biomass, characterized by its carbon neutrality. Because the carbon in biomass originates from atmospheric carbon dioxide fixed by plant photosynthesis, rather than from buried carbon in fossil fuels, carbon dioxide emissions during biomass-based hydrogen production are part of the natural cycle. Furthermore, as the world's fourth largest energy source, biomass resources are abundant and renewable, making it a highly promising source of green hydrogen energy. Utilizing biomass to produce hydrogen is an important way to address future low-carbon energy needs.

[0005] Starch (or its derivatives) is a low-cost and renewable carbohydrate with a high hydrogen density, and can be used to efficiently produce hydrogen through an in vitro multi-enzyme catalytic system.

[0006] However, in existing starch-based hydrogen production systems, the cofactor dependence of 6-phosphate gluconate dehydrogenase is NADP. + NADP + Cofactors are expensive, and large-scale addition is not permitted in practical industrial applications. Therefore, NADP could be considered as a potential alternative. + Replace with lower-cost NAD + However, this can easily introduce the effect of 6-phosphate gluconate dehydrogenase on coenzyme NAD. + A new problem has emerged: "low enzyme activity". Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide a novel NAD-dependent glucose-6-phosphate dehydrogenase and its application. This is achieved by mutating wild-type gluconeyl 6-phosphate dehydrogenase to obtain a novel NAD-dependent dehydrogenase. + A mutant with better enzyme activity, this mutant is an NAD-dependent glucose-6-phosphate dehydrogenase, which can be used for hydrogen production from starch, effectively reducing the cost of hydrogen production and facilitating industrial production.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0009] A novel NAD-dependent glucose-6-phosphate dehydrogenase, a mutant of 6-phosphate gluconate dehydrogenase, has the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.3. The coenzyme-binding specificity of this 6-phosphate gluconate dehydrogenase mutant undergoes a functional conversion, changing from wild-type dependence on nicotinamide adenine dinucleotide phosphate (NADP+ / NADPH) to selective dependence on nicotinamide adenine dinucleotide (NAD+ / NADH), thus enabling the binding of NAD+ to NADP+. + The activity is converted into NADH.

[0010] As one of the preferred embodiments of the present invention, it is obtained by mutating the wild-type 6-phosphoglucate dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1.

[0011] As one of the preferred embodiments of the present invention, the sequence corresponding to SEQ ID NO.2 is a single-point mutant R33V: the arginine at position 33 of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine.

[0012] As one of the preferred embodiments of the present invention, the sequence corresponding to SEQ ID NO.3 is a two-point mutant R33V / K37F: the arginine at position 33 of the amino acid sequence shown in SEQ ID NO.1 is mutated to valine, and the lysine at position 37 is mutated to phenylalanine.

[0013] The encoding gene of the novel NAD-dependent glucose-6-phosphate dehydrogenase described above has the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.5; wherein the sequence in SEQ ID NO.4 is used to encode the 6-phosphate gluconate dehydrogenase mutant corresponding to SEQ ID NO.2, and the sequence in SEQ ID NO.5 is used to encode the 6-phosphate gluconate dehydrogenase mutant corresponding to SEQ ID NO.3.

[0014] A recombinant expression vector containing the above-mentioned coding gene and a recombinant engineered bacterium.

[0015] A method for preparing the above-mentioned novel NAD-dependent glucose-6-phosphate dehydrogenase:

[0016] (1) The gene of the target enzyme shown in SEQ ID NO.4 or SEQ ID NO.5 is cloned into an expression vector and a 6xHis tag is fused to the C-terminus; then, the recombinant plasmid is introduced into a host strain to construct a recombinant engineered strain; wherein, the sequence of SEQ ID NO.4 is used to encode the 6-phosphoglucate dehydrogenase mutant corresponding to SEQ ID NO.2, and the sequence of SEQ ID NO.5 is used to encode the 6-phosphoglucate dehydrogenase mutant corresponding to SEQ ID NO.3;

[0017] (2) The recombinant engineered bacteria were cultured, and after inducing gene expression by adding IPTG, the cells were collected and subjected to ultrasonic lysis and centrifugation.

[0018] (3) The supernatant was further purified by heat treatment and the protein with the 6xHis tag was subjected to affinity chromatography using a Ni-NTA column;

[0019] (4) Remove impurities by dialysis and preserve the target protein.

[0020] Application of a novel NAD-dependent glucose-6-phosphate dehydrogenase in starch hydrogen production.

[0021] As one of the preferred embodiments of the present invention, the novel NAD-dependent glucose-6-phosphate dehydrogenase (a mutant of glucose-6-phosphate dehydrogenase) is introduced into a multi-enzyme reaction system for starch hydrogen production; the glucose-6-phosphate dehydrogenase is used to remove NAD from the multi-enzyme reaction system. + It is reduced to NADH, and the electron transfer between NADH and BV is catalyzed by electron mediators benzyl viologen (BV) and flavoxylase (DI). The biomimetic electron transfer chain from NADH to H2 is catalyzed by hydrogenase (SH I) to produce hydrogen gas.

[0022] As one of the preferred embodiments of the present invention, the multi-enzyme reaction system specifically includes HEPES buffer pH 7.5, maltodextrin (glucose equivalent 4-7), magnesium chloride, manganese chloride, NAD, benzyl violetin (BV), sodium phosphate, α-glucose transferase (αGP), phosphogluconomutase (PGM), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase mutant (6PGDH mutant), 6-phosphogluconolactonease (6PGL), flavoxelase (DI), hydrogenase (SHI), ribulose-5-phosphate-3-epimerase (RUPE), transketolase (TK), transaldolase (TAL), triose phosphate isomerase (TIM), fructose-1,6-bisphosphate aldolase (ALD), fructose-1,6-bisphosphatase (FBP), and phosphogluconomutase (PGI).

[0023] As one of the preferred embodiments of the present invention, the novel NAD-dependent glucose-6-phosphate dehydrogenase (a mutant of glucose-6-phosphate dehydrogenase) is added in the form of a pure enzyme solution.

[0024] A method for converting starch into hydrogen gas: A multi-enzyme reaction system is constructed in a reaction vessel, containing the following components: HEPES buffer pH 7.5, maltodextrin (glucose equivalent 4-7), magnesium chloride, manganese chloride, NAD, benzyl viologen (BV), sodium phosphate, α-glucose transferase (αGP), phosphogluconomutase (PGM), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase mutant (6PGDH mutant), 6-phosphogluconolactonease (6PGL), flavoxelase (DI), hydrogenase (SHI), ribulose-5-phosphate-3-epimerase (RUPE), transketolase (TK), transaldolase (TAL), triose phosphate isomerase (TIM), fructose-1,6-bisphosphatase (ALD), fructose-1,6-bisphosphatase (FBP), and phosphogluconomutase (PGI). The reaction system reacts to produce hydrogen gas.

[0025] As one of the preferred embodiments of the present invention, the reaction can be carried out at room temperature; however, it is more preferred to raise the reaction temperature to 80°C, as higher temperatures are more conducive to increasing the hydrogen production rate.

[0026] As one of the preferred embodiments of the present invention, the BV concentration in the reaction system is 2 mM.

[0027] As one of the preferred embodiments of the present invention, the NAD concentration in the reaction system is 8 mM.

[0028] The above method involves directly constructing a reaction system in a reaction vessel, utilizing maltodextrin (a starch derivative) to generate hydrogen gas under the action of fourteen enzymes.

[0029] The advantages of this invention compared to the prior art are:

[0030] This invention provides a technique for preparing hydrogen using maltodextrin (a starch derivative) as a substrate, achieving hydrogen production without the need for exogenous ATP through a multi-enzyme reaction system. Specifically, this invention utilizes an optimized and screened "6-phosphate gluconate dehydrogenase mutant" to replace the traditional NADP-dependent redox coenzyme system with a more economical and efficient NAD-dependent system. This key improvement significantly reduces production costs and simplifies reaction conditions. Compared to traditional microbial fermentation methods for hydrogen production (where 1 mol of glucose produces a maximum of 4 mol of hydrogen), this invention significantly increases hydrogen yield while extending the reaction time.

[0031] Furthermore, this invention employs multiple thermophilic enzymes as the core components of a multi-enzyme reaction system. This not only allows for convenient acquisition of the desired enzyme preparations through thermal purification but also enables further enhancement of the reaction rate by increasing the reaction temperature. More importantly, this system requires only the addition of a small amount of NAD to achieve the cyclic regeneration of NAD-NDH, avoiding dependence on the energy molecule ATP. This significantly reduces production costs and provides a feasible technical path for large-scale hydrogen production, facilitating industrial-scale manufacturing. Attached Figure Description

[0032] Figure 1 This is the protein content standard curve from Example 5;

[0033] Figure 2 This is the standard curve of NADH content in Example 5;

[0034] Figure 3 These are the enzyme activity results of wild-type Tne6PGDH and various mutants in Example 5;

[0035] Figure 4 This is a schematic diagram of the insertion site of the α-glucose transferase (αGP) gene in the corresponding vector in Example 6;

[0036] Figure 5 This is a schematic diagram of the insertion site of the phosphoglucosuric enzyme (PGM) gene in the corresponding vector in Example 6;

[0037] Figure 6 This is a schematic diagram of the insertion site of the glucose-6-phosphate dehydrogenase (G6PDH) gene in the corresponding vector in Example 6;

[0038] Figure 7 This is a schematic diagram of the insertion site of the 6-phosphogluconolactonease (6PGL) gene in the corresponding vector in Example 6;

[0039] Figure 8 This is a schematic diagram of the insertion site of the yellow transfectase (DI) gene in the corresponding vector in Example 6;

[0040] Figure 9 This is a schematic diagram of the insertion site of the hydrogenase (SHI) gene in the corresponding vector in Example 6;

[0041] Figure 10 This is a schematic diagram of the insertion site of the ribulose 5-phosphate 3-epimerase (RUPE) gene in the corresponding vector in Example 6;

[0042] Figure 11 This is a schematic diagram of the insertion site of the transketolase (TK) gene in the corresponding vector in Example 6;

[0043] Figure 12This is a schematic diagram of the insertion site of the transaldolase (TAL) gene in the corresponding vector in Example 6;

[0044] Figure 13 This is a schematic diagram of the insertion site of the triose phosphate isomerase (TIM) gene in the corresponding vector in Example 6;

[0045] Figure 14 This is a schematic diagram of the insertion site of the fructose-1,2-bisphosphate aldolase (ALD) gene in the corresponding vector in Example 6;

[0046] Figure 15 This is a schematic diagram of the insertion site of the fructose 1,6-bisphosphatase (FBP) gene in the corresponding vector of the present invention.

[0047] Figure 16 This is a schematic diagram of the insertion site of the phosphoglucose isomerase (PGI) gene in the corresponding vector in Example 6;

[0048] Figure 17 This is a comparison of hydrogen production rates of wild-type Tne6PGDH and mutants R33V and R33V / K37F in Example 7. Detailed Implementation

[0049] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, the carriers, reagents, and culture media used in the present invention, unless otherwise specified, are all conventional carriers, reagents, and culture media in this technical field. The experimental methods used in the present invention, unless otherwise specified, are all conventional methods in this technical field. For example, for gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al. The experimental equipment used in the present invention, unless otherwise specified, is all conventional equipment in this technical field and will not be described further.

[0050] The culture medium formulations involved in the following examples are as follows:

[0051] LB medium: a general-purpose commercial culture medium.

[0052] ASW-YT medium: Sodium chloride 3 g / L, sodium citrate 20 g / L, magnesium chloride hexahydrate 3 g / L, ammonium sulfate 1 g / L, potassium chloride 0.5 g / L, potassium dihydrogen phosphate 0.42 g / L, sodium bromide 0.05 g / L, strontium chloride hexahydrate 0.02 g / L, ferrous ammonium sulfate hexahydrate 0.01 g / L, cysteine ​​0.25 g / L, cysteine ​​hydrochloride monohydrate 0.25 g / L, MOPS 2 g / L, yeast extract 5 g / L, peptone 5 g / L, resazurin 0.001 g / L, magnesium sulfate heptahydrate 6 g / L, sodium bicarbonate 0.2 g / L, calcium chloride dihydrate 0.3 g / L, niacin 2 mg / L, biotin 0.8 mg / L, pantothenate 2 mg / L, lipoic acid 2 mg / L, Folicacid 0.8 mg / L, Aminobenzoic 2 mg / L, Thiamine 2 mg / L, Riboflavin 2 mg / L, Pyriodoxine 2 mg / L, Cobalamin 2 mg / L, Manganese sulfate monohydrate 2.5 mg / L, Cobalt chloride hexahydrate 0.5 mg / L, Zinc sulfate heptahydrate 0.5 mg / L, Copper sulfate pentahydrate 0.05 mg / L, Potassium aluminum sulfate dodecahydrate 0.05 mg / L, Boric acid 0.05 mg / L, Sodium molybdate dihydrate 0.05 mg / L, Nickel chloride hexahydrate 0.05 mg / L.

[0053] The buffer involved in the following embodiments:

[0054] Elution Buffer: 100mM PBS, NaCl 17.532g / L, imidazole 34.04g / L, pH 7.5.

[0055] Wash Buffer: 100mM PBS, NaCl 17.532g / L, imidazole 3.404g / L, pH 7.5.

[0056] Lysis Buffer: 100mM PBS, NaCl 17.532g / L, imidazole 0.68g / L, pH 7.5.

[0057] Example 1: Construction of engineered bacteria expressing wild-type 6-phosphate gluconate dehydrogenase (Tne6PGDH):

[0058] Plasmid Pet-28A was selected and digested with a restriction endonuclease (FastDigest Eco31I) and a wild-type 6-phosphate gluconate dehydrogenase Tne6PGDH derived from *Thermotoganeapolitana* (GenBank No.: KFZ22523.1, amino acid sequence as SEQ ID NO.1, nucleotide sequence as SEQ ID NO.6), which produced sticky ends at specific cleavage sites. DNA ligase was used to ligate the two, forming a complex. The ligated plasmid was transformed into Bl21(DE3) competent cells, incubated for 30 min, and then heat-shocked in 42°C water for 90 s. 600 μl of LB medium was added to the heat-shocked competent cells, and the cells were cultured in a shaker at 37°C for 1 h. After low-speed centrifugation, the supernatant was discarded, and 100 μl of the liquid was spread onto kanamycin-resistant culture dishes. After 14 h of culture, colonies were picked and cultured in test tubes for 8 h. Cell clones containing the target sequence were screened by culturing the transformed cells.

[0059] Example 2: Design of a 6-phosphate gluconate dehydrogenase mutant:

[0060] Based on the wild-type Tne6PGDH amino acid sequence (SEQ ID NO.1) of Example 1, the following mutation designs were carried out: (1) Single-point mutations were performed on positions 32, 33, and 34 of the SEQ ID NO.1 sequence; (2) Random mutations were performed simultaneously on positions 32, 33, and 34 of the SEQ ID NO.1 sequence; (3) After performing a single-point mutation on position 33 of the SEQ ID NO.1 sequence, mutations were then performed on positions 37, 75, and 100.

[0061] Primer sequences for PCR were designed for the mutants described above, as shown in Table 1.

[0062] Table 1 Primers designed for mutation sites.

[0063]

[0064] The PCR (25 μL) amplification system is as follows: 25 μL of 2×PCR buffer, 1.5 μL each of forward and reverse primers, 1 μL of template plasmid, 1 μL of dNTP, 1 μL of high-fidelity enzyme, and ddH2O added to make up to 50 μL.

[0065] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 5 min, 30 cycles, 72℃ extension for 10 min, and storage at 4℃.

[0066] After PCR, 5 μL of the amplified product was analyzed by nucleic acid gel electrophoresis. 2 μL of Dpn I restriction enzyme was added to the PCR product with a clear target band, and the sample was digested at 37°C for 1 hour. After the reaction, the product was cleaned up and transformed into BL21 competent cells, plated on LB solid medium containing 50 μg / mL kanamycin, and incubated overnight at 37°C. The cells were then harvested to obtain transformants containing the mutant.

[0067] Example 3: Initial screening of mutant engineered bacteria and acquisition of crude enzyme solution:

[0068] Transformants containing mutants obtained in Example 2 were evenly spread on 10 cm LB kanamycin agar plates (15 mL) and grown overnight at 37°C, with an expected colony count of 500 per plate. After incubation at room temperature for one day to overexpress the protein, the colonies were treated at 70°C for 1 hour to partially disrupt the cell membrane, inactivate the mesophilic host protein, and oxidize endogenous reducing compounds. The heat-treated colonies were then transferred to a 7.5 cm filter paper surface placed in a Buchner funnel. The colonies were then soaked in 50 mL of 50 mM sodium phosphate buffer (pH 7.5) for 3 minutes, followed by vacuum filtration to remove the buffer. The washing procedure was performed eight times to remove endogenous NADP from the colonies.

[0069] For the first round of screening of 6-phosphate gluconate dehydrogenase mutants, washed colonies were placed in new culture dishes containing 10 mL of 0.5% agarose gel with 150 μM WST-1, 0.13 μM GsDI (2.8 μg / mL), 2 mM substrate (such as 6PG or G6P), 1 mM NAD+, 50 mM tris-HCl (pH 7.5), 50 μg / mL chloramphenicol, and 0.1% sodium azide. The mixture was then covered with another 10 mL of the same 0.5% molten agarose solution (60°C). Screening for 6PGDH mutants was performed under light for 12 hours. To identify positive mutants through image analysis, colonies were photographed under white light with a consistent brightness level (colonies turning yellow were considered positive mutants). The following positive clones were finally obtained: N32E, R33V, T34S, T34I, N32E / T34K, N32E / T34G, N32E / T34A, N32E / T34C, N32E / T34L; R33V / K37F; R33V / K75C; and R33V / G100C.

[0070] Following the method described in Example 1, bacterial cells containing the positive mutant were obtained. The collected 6-phosphate gluconate dehydrogenase mutant was then washed twice with 50 mM pH 7.5 HEPES buffer, resuspended in 50 mL pH 7.5 HEPES buffer, homogenized, and centrifuged to remove the precipitate, yielding a crude enzyme solution containing the 6-phosphate gluconate dehydrogenase mutant. The crude enzyme solution of wild-type 6-phosphate gluconate dehydrogenase was obtained similarly.

[0071] Example 4, Protein Purification:

[0072] (1) Reagent preparation: Ni column equilibration buffer (1L): 50mM pH7 Tris-HCl, 200mM NaCl, 50mM imidazole, 2mM 2-mercaptoethanol; elution buffer (1L): 50mM pH7 Tris-HCl, 200mM NaCl, 250mM imidazole, 2mM 2-mercaptoethanol; 20% (v / v) ethanol (200mL).

[0073] (2) Sample processing: The crude enzyme solution of wild-type 6-phosphate gluconate dehydrogenase and its mutant obtained in the above examples was used as a protein purification sample.

[0074] (3) Rinse the protein purification instrument tubing with ultrapure water at a flow rate of 4 mL / min for 10 min.

[0075] (4) Connect the Ni column to the instrument and flush with equilibration solution at a flow rate of 4 mL / min until the baseline is stable.

[0076] (5) Load the sample at a flow rate of 2 mL / min, with a single loading volume of approximately 20 mL. Observe the remaining sample volume and avoid inhaling air.

[0077] (6) After loading the sample, continue to rinse with equilibration buffer at a flow rate of 2 mL / min until the baseline is stable.

[0078] (7) After the baseline stabilizes, rinse with elution buffer at a flow rate of 2 mL / min and observe the changes in UV value. When the UV value shows an upward peak, collect the protein in a pre-cooled 10 mL centrifuge tube after 90 s and place it on ice.

[0079] (8) Continue rinsing with elution buffer until the baseline is stable.

[0080] (9) Rinse with equilibration solution to stabilize the baseline again, and continue with the next sample loading and purification.

[0081] (10) After the end, flush the column with 20% ethanol at a flow rate of 5 mL / min for 20 min to preserve the column.

[0082] The entire purification process must be kept at a low temperature to avoid enzyme inactivation.

[0083] Example 5: Enzyme activity of 6-phosphoglucose dehydrogenase and its mutants:

[0084] The positive clones N32E, R33V, T34S, T34I, N32E / T34K, N32E / T34G, N32E / T34A, N32E / T34C, N32E / T34L, R33V / K37F, R33V / K75C, and R33V / G100C obtained from the initial screening and after protein purification were re-screened (by detecting the catalytic efficiency of the mutants by HPLC).

[0085] Specifically, the protein concentration of the purified wild-type 6-phosphate gluconate dehydrogenase and its positive clones was determined using a BCA kit. The principle is as follows: Cu 2+ It is reduced to Cu in a slightly alkaline environment. 2+ It can form a blue complex with BCA reagent, exhibiting a specific absorbance at 562 nm. The protein content can then be calculated by referring to a standard curve. The specific operating procedure is as follows:

[0086] I. Plotting the standard curve.

[0087] (1) Add reagents to a clean standard 96-well transparent plate according to Table 2.

[0088] Table 2. Reagent Addition for Transparent Plates

[0089] Kong Hao 1 2 3 4 5 6 7 8 Protein standard solution (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 Corresponding protein content (μg) 0 0.5 1.0 2.0 4.0 6.0 8.0 10.0

[0090] (2) Preparation of BCA working solution

[0091] Add BCA "Reagent A" to BCA "Reagent B" in a volume ratio of "50:1" into a 2mL EP tube, mix well, and set aside.

[0092] (3) Add the prepared working solution to the first 8 wells, immediately place in the microplate reader, gently shake, incubate at 37°C for 30 min, and then measure the absorbance at 562 nm. A protein content standard curve can be plotted based on the absorbance values ​​corresponding to the standard protein concentrations. Figure 1 The standard curve equation is y = 0.19397x + 0.03427.

[0093] (4) The catalytic efficiency of 6-phosphate gluconate dehydrogenase and its mutant was compared by measuring the amount of NADH produced by absorbance. Enzyme activity is defined as the amount of enzyme required to catalyze the reduction of NAD to 1 μmol of NADH per minute at 35℃ and pH 7.5. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the reduction of NAD to 1 μmol of NADH per minute.

[0094] Specifically, the reaction system (1 mL) is prepared as follows: 100 mM pH 7.5 HEPES, 1 mM NAD, 2 mM glucose 6-phosphate, 5 mM MgCl2, 0.5 mM MnCl2, 0.1 g / L gluconate 6-phosphate dehydrogenase or its mutant purified enzyme.

[0095] After the reaction system is prepared, mix well and react for 10 minutes. Calculate enzyme activity by detecting the amount of NADH produced using an enzyme-linked immunosorbent assay (ELISA) reader, and then analyze the NADH content standard curve. Figure 2 The standard curve equation (y = 28.128x + 0.1802) was used to obtain the protein concentrations of 6-phosphate gluconate dehydrogenase and its mutants, thereby calculating the specific enzyme activity of 6-phosphate gluconate dehydrogenase and its mutants. Each sample was tested in triplicate.

[0096] The final enzyme activity results are shown in Table 3 and Figure 3 As shown.

[0097] Table 3 Enzyme activity results

[0098]

[0099]

[0100] The results show that the enzyme activity of R33V is significantly increased in the single-point mutant and the enzyme activity of R33V / K37F is significantly increased in the double-point mutant.

[0101] Accordingly, mutants R33V and R33V / K37F can be selected for subsequent starch hydrogen production. The specific amino acid sequence of the single-point mutant R33V is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.4. The amino acid sequence of the double-point mutant R33V / K37F is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.5.

[0102] Example 6: Construction of a multi-enzyme reaction system:

[0103] The multi-enzyme reaction system for starch hydrogen production of this invention involves α-glucose transferase (αGP), phosphogluconomutase (PGM), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), 6-phosphogluconolactonease (6PGL), flavotransferase (DI), hydrogenase (SHI), ribulose-5-phosphate-3-epimerase (RUPE), transketolase (TK), transaldolase (TAL), triose phosphate isomerase (TIM), fructose-1,6-bisphosphatase (ALD), fructose-1,6-bisphosphatase (FBP), and phosphogluconomutase (PGI).

[0104] Among them, 6-phosphoglucate dehydrogenase (6PGDH) was wild-type Tne6PGDH or the mutant R33V or R33V / K37F finally screened in this invention. The other enzymes were existing enzyme preparations, such as αGP, PGM, G6PDH, 6PGL, DI, SHI, RUPE, TK, TAL, TIM, ALD, FBP, and PGI, all of which were reported in the article "Ultra-rapid rates of water splitting for biohydrogen gas production through in vitro artificial enzymatic pathways".

[0105] The aforementioned existing enzyme preparations can also be obtained using the following methods:

[0106] (1) Referring to Table 4, insert the target enzyme gene into the corresponding vector to construct a recombinant plasmid; and transfer the recombinant plasmid to the host bacteria to construct a recombinant engineered bacteria (this step is handled by a biotechnology company).

[0107] Table 4 Construction of recombinant engineered bacteria

[0108]

[0109]

[0110] (2) Expression and purification of αGP, PGM, G6PDH, 6PGL, DI, SHI, RUPE, TK, TAL, TIM, ALD, FBP, and PGI proteins:

[0111] Recombinant engineered bacteria expressing αGP, PGM, G6PDH, 6PGL, DI, RUPE, TK, TAL, TIM, ALD, FBP, and PGI proteins were cultured in LB medium. The bacterial cells were then collected, resuspended in Lysis Buffer, and disrupted (ultrasonic disruption / high-pressure homogenization). The cells were centrifuged at 12000 rpm for 1 hour. The supernatant was poured into a nickel column, and the recyclable liquid was repeated twice. Washing with Wash Buffer was added to remove contaminating proteins, followed by a small amount of Elution Buffer. Two or three elutions were usually sufficient to remove most of the protein. These protein solutions were collected in ultrafiltration tubes for desalting. Ultrafiltration reduced the concentrations of imidazole and sodium chloride to below 1 mM. The resulting enzyme solution was then diluted with 10% glycerol, and the protein concentration was measured. The solution was then stored at -80°C for later use.

[0112] The recombinant engineered bacteria corresponding to SHI were cultured in ASW-YT medium, and then the bacterial cells were collected and purified in an anaerobic chamber. The purification process was the same as that for the other enzyme proteins mentioned above. The resulting enzyme solution was added to a final concentration of 10% glycerol, the protein concentration was detected, and then it was stored in a -80℃ freezer for later use.

[0113] Accordingly, the multi-enzyme reaction system (5 mL) of the present invention was constructed:

[0114] HEPES buffer (pH 7.5) 0.1M, maltodextrin (glucose equivalent 4-7) 0.082M, magnesium chloride 5mM, manganese chloride 0.5mM, NAD 8mM, benzyl viologen (BV) 2mM, 50mM sodium phosphate, α-glucose transferase (αGP) 0.625mg / mL, phosphogluconomutase (PGM) 0.125mg / mL, glucose-6-phosphate dehydrogenase (G6PDH) 1.042mg / mL, phosphogluconate dehydrogenase (wild-type Tne6PGDH, mutant R33V, or mutant R33V / K37F) 0.357mg / mL, phosphogluconolactone (6PGL) 0.054mg / mL, flavoxelase (DI) 2. 5 mg / mL, hydrogenase (SHI) 2.5 mg / mL, ribulose 5-phosphate 3-epimerase (RUPE) 0.075 mg / mL, transketolase (TK) 0.943 mg / mL, transaldolase (TAL) 1.282 mg / mL, triose phosphate isomerase (TIM) 0.0055 mg / mL, fructose diphosphate aldolase (ALD) 0.234 mg / mL, fructose 1,6-bisphosphatase (FBP) 0.833 mg / mL, glucose phosphate isomerase (PGI) 0.0658 mg / mL.

[0115] Example 7: Verification of catalytic efficiency:

[0116] The multi-enzyme reaction system shown in Example 6 was constructed in a reactor and reacted at 80°C. Nitrogen gas at a flow rate of 50 mL / min was used as the carrier gas to carry the reaction gas out of the reaction vessel and pass it into a hydrogen analyzer for detection.

[0117] The final result is as follows Figure 17 As shown in the figure. The results above indicate that the hydrogen production rate of the mutant R33V / K37F is significantly higher than that of the wild-type Tne6PGDH (WT Tne6PGDH) and the single-point mutant R33V.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel NAD-dependent glucose-6-phosphate dehydrogenase, characterized in that, It is a mutant of 6-phosphoglucate dehydrogenase, and its amino acid sequence is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. A gene encoding a novel NAD-dependent glucose-6-phosphate dehydrogenase as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.4 or SEQ ID NO.5; wherein, the sequence of SEQ ID NO.4 is used to encode the 6-phosphoglucate dehydrogenase mutant corresponding to SEQ ID NO.2, and the sequence of SEQ ID NO.5 is used to encode the 6-phosphoglucate dehydrogenase mutant corresponding to SEQ ID NO.

3.

3. A recombinant expression vector or recombinant engineered bacteria containing the encoding gene as described in claim 2.

4. A method for preparing the novel NAD-dependent glucose-6-phosphate dehydrogenase as described in claim 1, characterized in that, Includes the following steps: (1) The gene of the target enzyme shown in SEQ ID NO.4 or SEQ ID NO.5 is cloned into an expression vector and a 6xHis tag is fused to the C-terminus; then, the recombinant plasmid is introduced into the host strain to construct a recombinant engineered strain; wherein, the sequence of SEQ ID NO.4 is used to encode the 6-phosphate gluconate dehydrogenase mutant corresponding to SEQ ID NO.2, and the sequence of SEQ ID NO.5 is used to encode the 6-phosphate gluconate dehydrogenase mutant corresponding to SEQ ID NO.3; (2) The recombinant engineered bacteria were cultured, and after inducing gene expression by adding IPTG, the cells were collected and subjected to ultrasonic lysis and centrifugation. (3) The supernatant was further purified by heat treatment and the protein with the 6xHis tag was subjected to affinity chromatography using a Ni-NTA column; (4) Remove impurities by dialysis and preserve the target protein.

5. The application of a novel NAD-dependent glucose-6-phosphate dehydrogenase as described in claim 1 in starch hydrogen production, characterized in that, The novel NAD-dependent glucose-6-phosphate dehydrogenase was introduced into a multi-enzyme reaction system for starch hydrogen production; the glucose-6-phosphate dehydrogenase was used to remove NAD from the multi-enzyme reaction system. + It is reduced to NADH, and electron transfer between NADH and BV is achieved through the electron mediators benzyl viologen (BV) and flavoxylase DI, where DI is the catalyst and BV is the electron mediator. Then, through the biomimetic electron transfer chain from NADH to H2, combined with the catalysis of hydrogenase SHI, hydrogen gas is produced.

6. The application according to claim 5, characterized in that, The novel NAD-dependent glucose-6-phosphate dehydrogenase was added in the form of a pure enzyme solution.

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