A coenzyme q10 reductase mutant, carrier, strain and application thereof

Abstract

The application provides a coenzyme Q10 reductase mutant, a carrier, a strain and application thereof. The coenzyme Q10 reductase mutant is mutated at multiple amino acid sites in the amino acid sequence shown in SEQ ID NO. 1. By using the coenzyme Q10 reductase mutant of the application, oxidized coenzyme Q10 and NADH are used as raw materials, and through the oxidation-reduction reaction of the enzyme, a cyclic regeneration reaction of NAD+ to NADH is carried out by using formate dehydrogenase, and reduced coenzyme Q10 is efficiently prepared. Compared with the enzyme before mutation, the mutant enzyme has higher catalytic activity and conversion yield, and is more suitable for large-scale industrial production application.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a coenzyme Q10 reductase mutant, vector, strain and their applications. Background Technology

[0002] Coenzyme Q10 is a vitamin-like substance found in the mitochondria of various animal, plant, and microbial cells. It is a naturally synthesized antioxidant and cell metabolism activator, closely related to respiration, and plays a vital role in the transmission of cellular electron signals and the production of ATP. Coenzyme Q10 has a clear therapeutic effect on many diseases and can be used to treat cardiovascular diseases, Parkinson's disease, Alzheimer's disease, chronic obstructive pulmonary disease, AIDS (adjunctive therapy), male infertility, epilepsy, and insulin resistance syndrome (Syndrome X). It also has the effects of scavenging oxygen free radicals and anti-oxidation, which can delay skin aging and resist radiation. Therefore, it is added to many daily chemical products and health supplements and is popular among consumers. In China, Coenzyme Q10 is also beginning to enter the food industry, used as a food supplement, and can be added to edible oils and beverages, providing certain health benefits.

[0003] Currently, coenzyme Q10 is produced through chemical synthesis, biological tissue extraction, plant cell culture, and microbial fermentation. Microbial fermentation is the primary industrial method. It offers advantages such as a wide availability of raw materials, low cost, and the ability to produce on a high-density, large-scale basis. However, its post-fermentation purification technology still needs improvement, and the coenzyme Q10 extracted through fermentation is in its oxidized form. In recent years, reduced coenzyme Q10 has received increasing attention due to its advantages over oxidized coenzyme Q10: 1. Reduced coenzyme Q10 has a higher absorption rate in the human body than oxidized coenzyme Q10; 2. Reduced coenzyme Q10 can increase cellular energy, as the oxidized form must be reduced to its reduced form in cells to exert its antioxidant effect; 3. Reduced coenzyme Q10 can be used to prepare more novel dosage forms, such as injections; 4. Reduced coenzyme Q10 has higher bioavailability in the human body and can be applied in functional foods, health foods, nutritional supplements, beverages, cosmetics, and other fields. Currently reported methods for producing reduced coenzyme Q10 are mainly chemical methods, such as Chinese patent CN1551864A(WO2003 / 006412): Preparation method of reduced coenzyme Q10, Chinese patent CN1551863A(WO2003 / 006410): Preparation method of oily product of reduced coenzyme Q10, Chinese patent CN1527807A(WO2003 / 006408): Production method of reduced coenzyme Q10 using solvent with high antioxidant effect, and Chinese patent CN1723181A(WO2004 / 063131): Purification method of reduced coenzyme Q10, etc. The common drawback of these methods is that chemical reagents are required during the reaction, and these chemical reagents need to be removed through complex purification processes, resulting in problems such as complex production processes, high costs, and poor product stability.

[0004] To address the problems arising from the aforementioned chemical production methods, enzymatic catalysis is used to produce reduced coenzyme Q10. Figure 1 This method has become the preferred method for most manufacturers. To date, there are few reports on the production of reduced coenzyme Q10 using the biological enzymatic method. Only patents CN101429530A and CN101307338B report on the process research of producing reduced coenzyme Q10 using the biological enzymatic method. The former has not been authorized, and the latter only uses wild-type strains containing reductase for transformation production. It requires the preparation of phosphorylated oxidized coenzyme Q10 and the extraction of low-content oxidoreductase, which makes the production process complex and costly.

[0005] To address the high cost of enzymatic production of reduced coenzyme Q10, we started with the yeast-derived coenzyme Q10 reductase ScQ10R. Through the construction of mutant libraries and targeted screening, we obtained a series of mutant enzymes with high activity and high conversion rate (the optimal mutant showed a 7-fold increase in activity and a 10% increase in conversion rate), laying the foundation for the industrial application of enzymatic production of reduced coenzyme Q10. Summary of the Invention

[0006] The primary objective of this invention is to provide a coenzyme Q10 reductase mutant that exhibits higher catalytic activity compared to the wild-type ScQ10R, and a higher conversion yield when using oxidized coenzyme Q10 and NADH as raw materials to catalyze the preparation of reduced coenzyme Q10.

[0007] To achieve this objective, in a basic embodiment, the present invention provides a coenzyme Q10 reductase mutant, wherein the mutant mutates multiple amino acid sites in the coenzyme Q10 reductase ScQ10R of the amino acid sequence shown in SEQ ID NO.1, and the mutated amino acid sites are one or both of F54L and G106S.

[0008] Furthermore, the mutation mode is either one of the following two: G106S; F54L / G106S, preferably F54L / G106S.

[0009] A second objective of this invention is to provide a polynucleotide encoding the aforementioned coenzyme Q10 reductase mutant, such that the encoded coenzyme Q10 reductase mutant exhibits higher catalytic activity and conversion yield than the wild-type coenzyme Q10 reductase ScQ10R.

[0010] A third objective of this invention is to provide a vector containing the aforementioned coenzyme Q10 reductase mutant polynucleotide.

[0011] A fourth objective of this invention is to provide engineered microorganisms or cells, particularly strains, containing the aforementioned carriers.

[0012] A fifth object of this invention is to provide an application of the aforementioned coenzyme Q10 reductase mutant for the preparation of reduced coenzyme Q10, thereby enabling the better preparation of reduced coenzyme Q10 products.

[0013] Specifically, oxidized coenzyme Q10 and NADH are used as raw materials. Under the action of the coenzyme Q10 reductase mutant, formate dehydrogenase is used to catalyze the NADH recycling reaction to prepare reduced coenzyme Q10.

[0014] The reaction parameters are as follows: reaction temperature 25-35℃, substrate oxidized coenzyme Q10 concentration 10-15mM, NADH concentration 2-5mM, ammonium formate concentration 20-30mM, sodium sulfite concentration 10-20mM; substrate solvent 0.5-0.1mol / L phosphate buffer, reaction pH 6.5-7.0, reaction time 2-6h, formate dehydrogenase activity 70-80U, coenzyme Q10 reductase mutant 500-600U, and total reaction volume 100ml.

[0015] A sixth objective of this invention is to provide the application of the aforementioned carrier, or the aforementioned engineered microorganism or cell, in the preparation of coenzyme Q10 reductase, or the synthesis of reduced coenzyme Q10. The beneficial effects of this invention are that, when using the coenzyme Q10 reductase mutant of this invention to prepare reduced coenzyme Q10, compared with the enzyme before mutation, it exhibits higher catalytic activity and conversion yield, reduces production costs, and is more suitable for industrial-scale production applications. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the principle of coenzyme Q10 reductase catalyzing the production of reduced coenzyme Q10.

[0017] Figure 2 HPLC detection results for the preparation of reduced coenzyme Q10 catalyzed by wild-type ScQ10R.

[0018] Figure 3 HPLC detection results for the preparation of reduced coenzyme Q10 catalyzed by the ScQ10R-3 mutant. Detailed Implementation

[0019] The specific embodiments of the present invention will be further described below with reference to the examples and accompanying drawings.

[0020] The methods for detecting the enzyme activity of coenzyme Q10 reductase and its mutants are as follows:

[0021] Accurately transfer 0.5 ml of sample (diluted with water according to the concentration factor) into a 1.5 ml centrifuge tube (pre-fill the centrifuge tube with a certain amount of glass beads, preferably to 2 / 3 of the tube height), and shake on an MS3 shaker for 20 min (shaker frequency set to 1500 / min). Pour the sample along with the glass beads into a beaker, add a certain amount of 0.87% physiological saline to dilute to an enzyme activity of 0.8–6.5 u / ml, centrifuge, and collect the supernatant as the crude enzyme solution.

[0022] In a 1 cm quartz cuvette, add 3.2 ml of 0.1 mol / L, pH 7.0 phosphate buffer (preheated to 30°C) and 100 μl of 50 mmol / L NADH solution, followed by 100 μl of crude enzyme solution. Mix thoroughly and zero the sample at 420 nm. Add 100 μl of 50 mmol / L potassium ferricyanide solution (preheated to 30°C), mix quickly, and begin measurement. Record the absorbance A420 every 10 seconds for 10 measurement points. Calculate the enzyme activity by substituting the reaction rate and the molar extinction coefficient of potassium ferricyanide measured at this wavelength into the enzyme activity formula.

[0023] Substrate preparation:

[0024] 50 mmol / L potassium ferricyanide solution: Weigh 0.16 g of potassium ferricyanide, dissolve it in 0.1 mol / L pH 7.0 phosphate buffer and bring the volume up to 10 ml.

[0025] 50 mmol / L NADH solution: Weigh 354.5 mg of NADH, dissolve it in deionized water and bring the volume to 10 ml.

[0026] Enzyme activity calculation:

[0027] Liquid enzyme activity at 30℃ = [ΔA / min] * [1 / S] * [1 / d] * [Vt / Vs] * X (u / ml)

[0028] Enzyme activity unit: Under certain reaction conditions, one unit is defined as the amount of enzyme that generates or consumes 1 μmol of NADH per minute.

[0029] ΔA / min — represents the change in absorbance per minute, which is the slope;

[0030] The molar extinction coefficient of S-potassium ferricyanide was obtained through a standard curve.

[0031] d—optical path length of the cuvette (1 cm);

[0032] Vt — represents the total volume of the reaction solution, in ml.

[0033] Vs—indicates the volume of the sample enzyme solution, in ml;

[0034] X—indicates the dilution factor of the sample enzyme solution;

[0035] Example 1: Construction and screening of a library of error-prone mutants of coenzyme Q10 reductase ScQ10R

[0036] The coenzyme Q10 reductase ScQ10R in this invention is derived from yeast, and its amino acid sequence is shown in SEQ ID NO.1. The codons encoding the above ScQ10R amino acid sequence were artificially optimized, and the optimized polynucleotide sequence is shown in SEQ ID NO.2. This polynucleotide sequence was then artificially synthesized and cloned into the NdeI and XhoI multiple cloning sites of the expression vector pET30a(+) to obtain the recombinant expression plasmid pET30-ScQ10R, which was then transformed into Escherichia coli BL21(DE3) for expression.

[0037] Escherichia coli BL21(DE3) / pET30-ScQ10R was inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C at 220 rpm. Bacterial cells were collected, and plasmids were extracted using a plasmid kit (OMEGA). Using plasmid pET30-ScQ10R as a template, a fault-prone mutation library was constructed. The specific construction procedure is as follows: Using the extracted plasmid as a template, conventional universal primers (universal primer sequences: T7F: 5'-TAATACGACTCACTATAGGG-3'; T7R: 5'-GCTAGTTATTGCTCAGCGG-3') were selected for fault-prone PCR. The Mg content in the PCR amplification reaction system was adjusted. 2+ Mn 2+ The concentrations of dCTP and dTTP oligonucleotides were adjusted to ensure that the base mismatch rate of the mutant library was only 0.2%, guaranteeing that only 1 to 2 amino acids were mutated in each mutant. The error-prone PCR reaction system and procedure are as follows:

[0038] Error-prone PCR reaction system:

[0039]

[0040]

[0041] Error-prone PCR reaction procedure: First, pre-denaturate at 95℃ for 5 min; then denature at 95℃ for 30 s, anneal at 56℃ for 30 s, extend at 72℃ for 1 min, for a total of 30-35 cycles; finally, extend at 72℃ for 10 min. Take 2 μL of the above error-prone PCR product for agarose gel electrophoresis. After successful detection, purify and recover the PCR product using a DNA product purification kit. At 37℃, double digest the purified PCR product and the prokaryotic expression vector pET30a(+) with NdeI and XhoI restriction endonucleases, respectively. The digested products are then gel-recovered (the recovered purified PCR product fragment is approximately 900 bp, and the recovered pET30a(+) vector fragment is approximately 5400 bp). Mix the error-prone PCR product and the prokaryotic expression vector pET30a(+) at a molar ratio of 3:1, add T4 DNA ligase, and incubate overnight at 16℃. The following day, the ligation product was purified and recovered using a DNA product purification and recovery kit. The recovered product was used for electroporation transformation of Escherichia coli BL21(DE3). After electroporation, an appropriate amount of bacterial cells was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. The single colonies formed on the plates constituted the error-prone mutant library of ScQ10R.

[0042] Example 2: High-throughput screening of ScQ10R error-prone mutant libraries

[0043] Using sterilized toothpicks, carefully pick single colonies from the mutant library (one colony per toothpick) and inoculate them into different wells of a 96-well cell culture plate (each well already containing LB liquid medium with 50 μg / mL kanamycin). Incubate the 96-well cell culture plate at 37°C and 700 rpm for 6 hours on a constant-temperature high-speed shaker. Transfer 50 μL of cell culture medium from the plate to a sterile 96-well microplate as seed culture. Then, using an 8-channel pipette, add lactose to the 96-well plate to a final concentration of 1% (m / v) and induce incubation at 25°C and 700 rpm for 8 hours. After induction, freeze the 96-well cell culture plate at -80°C for 2 hours, then allow it to stand at room temperature for half an hour. Centrifuge at 4000 rpm and 4°C for 10 minutes, and transfer 50 μL of supernatant from each well to a 96-well microplate for subsequent high-throughput screening reactions. Preparation of reagents for high-throughput screening reactions:

[0044] Substrate solution: Dissolve 1g of oxidized coenzyme Q10 and 2g of NADH in 100ml of 0.1mol / L, pH 7.0 phosphate buffer by stirring thoroughly.

[0045] High-throughput screening colorimetric reaction:

[0046] In a 96-well microplate containing 50 μL / well of lysed bacterial cell supernatant, 50 μL / well of reaction substrate solution was added. The absorbance at OD320 nm was measured using a microplate reader and recorded as data A. After incubation at 30°C for 60 min, the absorbance at OD320 nm was measured again and recorded as data B. The decrease in absorbance from A to B was analyzed, and wells with the larger difference between A and B were selected for subsequent shake-flask fermentation, sequencing analysis, and transformation validation.

[0047] After multiple rounds of mutant library construction and screening, approximately 20,000 clones were selected. Following repeated verification through primary and secondary screening processes, strains with large absorbance differences were chosen for shake-flask fermentation and activity analysis. This yielded two high-activity strains expressing coenzyme Q10 reductase. Subsequent sequencing analysis revealed two mutant enzymes, as shown in Table 1.

[0048] Table 1: High-activity ScQ10R mutants obtained through screening

[0049]

[0050] As shown in the table above, the two mutant strains ScQ10R-1 and ScQ10R-2 obtained through screening exhibited fermentation activity increases ranging from 160% to 300% compared to the wild-type ScQ10R. The next step is to construct double-mutant enzymes based on the two favorable mutation points mentioned above, using the wild-type ScQ10R as a foundation.

[0051] Example 3: Construction and screening of ScQ10R site-directed superposition mutants

[0052] Starting with the strain expressing ScQ10R-1, after shake-flask culture and plasmid extraction, the G106S mutation site was superimposed to obtain the expression strain ScQ10R-3 containing both F54L and G106S double mutation sites. After shake-flask fermentation, sequencing analysis, and activity assay screening, the mutant enzyme ScQ10R-3 with enhanced activity was finally obtained, as shown in Table 2:

[0053] Table 2: ScQ10R mutants obtained by superposition mutation construction

[0054]

[0055] As shown in Table 2, the double mutant ScQ10R-3, which was generated by superimposing the beneficial mutation sites F54L and G106S, showed a significantly increased activity compared to the wild type, with an increase of up to 700%. To further verify the catalytic performance of the mutant enzyme, we selected the expression strain of the mutant ScQ10R-3 with the highest activity for fermentation culture, cell collection, and subsequent catalytic reduction and transformation reaction.

[0056] Example 4: Transformation Application of ScQ10R-3 Mutant Enzyme

[0057] Recombinant engineered strains expressing wild-type ScQ10R and the mutant ScQ10R-3 were inoculated separately into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. The inoculum was then transferred at a rate of 2% (v / v) to shake flasks containing 500 mL of TB liquid fermentation medium and cultured at 37°C and 220 rpm for 6 h. Lactose was added to a final concentration of 1% (m / v), and the mixture was cooled to 25°C for induction culture for 8 h. The entire fermentation broth was collected by centrifugation at 4°C and 10,000 rpm. The cells were washed twice with phosphate buffer (pH 7.0, 0.1 mol / L), centrifuged again, and concentrated 5-fold before resuspending in 100 mL of phosphate buffer (pH 7.0, 0.1 mol / L). The concentrated bacterial solution was then subjected to ultrasonic disruption in ice water until clear. The ultrasonic disruption conditions were: 3 seconds of operation, 5 seconds of interval, 30 cycles, and an ultrasonic power of 500 W.

[0058] Using the ultrasonically disrupted bacterial broths containing wild-type ScQ10R and ScQ10R-3 mutant enzymes, catalytic reduction reactions were carried out with oxidized coenzyme Q10 and NADH as substrates, respectively. The reaction conditions were as follows: In a 100 ml reaction system, a substrate solution containing 12.5 mM (1.08 g) oxidized coenzyme Q10, 3 mM (0.2 g) NADH, 27.5 mM (0.17 g) ammonium formate, and 16 mM (0.2 g) sodium sulfite was prepared in pH 7.0, 0.1 mol / L phosphate buffer. Then, 520 U of wild-type ScQ10R or ScQ10R-3 mutant enzyme and 72 U of formate dehydrogenase were added sequentially, and the reaction was carried out at 30 °C for 4 h. The target product, reduced coenzyme Q10, was detected by high-performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: column: C18; mobile phase: methanol:ethanol = 1:1; flow rate: 1 ml / min; wavelength: 275 nm; sample loading: 20 μL; detection temperature: 30℃. Weigh 5 mg of reduced coenzyme Q10, dissolve it in ethanol to 10 ml, inject 20 μL, and the peak elution time was approximately 11 min.

[0059] The reaction was monitored using HPLC. After the reaction was completed, the yield of reduced coenzyme Q10 in each reaction solution was analyzed, and the conversion yield (molar ratio of reduced coenzyme Q10 to substrate oxidized coenzyme Q10) was calculated. The final conversion results are shown in Table 3, and the HPLC chromatograms are shown in the figure below. Figure 2 and Figure 3 As shown.

[0060] Table 3: Yield analysis of ScQ10R mutant after reaction completion

[0061]

[0062] As can be seen from Table 3, under the same reaction conditions and the same amount of enzyme, the transformation completion time of wild-type ScQ10R and mutant ScQ10R-3 is comparable. However, the transformation yield of the mutant (97.07%) is about 10% higher than that of the wild-type (86.32%), which greatly reduces the production cost of reduced coenzyme Q10 and is suitable for large-scale industrial production.

[0063] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

[0064] SEQ ID NO.1 Wild-type ScQ10R amino acid sequence from yeast.

[0065] MSKEDIEGTNILDEPVHGIYIPAALFVVGVAITTYMSGELKILWSLPILFIIIFVRTYTA

[0066] YKRRRSLYPDRWTALELEDQTIISKNTALYRFKLKTRLESLDIPAGHHIAVRVPIDGK

[0067] EEVRYYNPISSKLESGYLDLVVKAYADGKVSKYFAGLNPGDTVDFKGPIGTLNYEPN

[0068] SSKHLGIVAGGSGITPVLQILNEIITVPEDLTKVSLLYANETENDILLKDELDEMAEKY

[0069] PHFQVHYVVHYPSDRWTGDVGYITKDQMNRYLPEYSEDNRLLICGPDGMNNLALQ

[0070] YAKELGWKVNSTRSSGDDQVFVF

[0071] SEQ ID NO.2 Wild-type ScQ10R nucleotide artificial sequence from the genus Saccharomyces

[0072] ATGAGCAAAGAAGATATCGAAGGTACAAATATTCTGGATGAACCGGTTCA

[0073] TGGCATCTATATTCCGGCAGCACTGTTTGTGGTGGGCGTTGCAATTACCAC

[0074] CTATATGAGCGGCGAACTGAAAATTCTGTGGAGCCTGCCGATTCTGTTTAT

[0075] TATTATTTTTGTGCGCACCTATACCGCATATAAACGTCGTCGCAGCCTGTA

[0076] TCCGGATCGCTGGACCGCCCTGGAACTGGAAGATCAGACCATTATTAGCA

[0077] AAAATACCGCACTGTATCGTTTTAAACTGAAAACCCGCCTGGAAAGTCTG

[0078] GATATTCCGGCAGGTCATCATATTGCAGTGCGTGTGCCGATTGATGGTAAA

[0079] GAAGAAGTTCGTTATTACAATCCGATTAGCAGTAAACTGGAAAGTGGCTA

[0080] TCTGGATCTGGTGGTTAAAGCCTATGCCGATGGCAAAGTGAGTAAATATTT

[0081] TGCCGGTCTGAATCCGGGCGATACCGTGGATTTTAAAGGCCCGATTGGTAC

[0082] ACTGAATTATGAACCGAATAGTAGCAAACATCTGGGCATTGTTGCCGGTG

[0083] GTAGCGGTATTACCCCGGTTCTGCAGATTCTGAATGAAATTATTACCGTGC

[0084] CGGAAGATCTGACCAAAGTTAGTCTGCTGTATGCAAATGAAACCGAAAAT

[0085] GATATTCTGCTGAAAGATGAACTGGATGAAATGGCAGAAAAATATCCGCA

[0086] TTTTCAGGTGCATTATGTGGTTCATTATCCGAGTGATCGTTGGACCGGTGA

[0087] CGTGGGCTATTACCAAAGATCAGATGAATCGTTACCTGCCGGAATATA

[0088] GCGAAGATAATCGCCTGCTGATTTGCGGCCCGGATGGTATGAATAATCTG

[0089] GCCCTTGCAGTATGCCAAAGAACTGGGTTGGAAAGTGAATAGCACCCGCAG

[0090] TAGTGGCGATGATCAGGTGTTTGTTTTT。

Claims

1. A mutant of a coenzyme Q10 reductase, characterized in that: Mutations are made in the amino acid sequence shown in SEQ ID NO.1, with the mutated amino acid sites being one or both of F54L and G106S.

2. The mutant according to claim 1, characterized in that: The mutation can be either one of the following two: G106S or F54L / G106S double mutation.

3. The mutant according to claim 2, characterized in that: The mutation mode is F54L / G106S double mutation.

4. A polynucleotide encoding a coenzyme Q10 reductase mutant according to any one of claims 1-3.

5. A vector containing a polynucleotide of the coenzyme Q10 reductase mutant as described in claim 4.

6. Engineered microorganisms or cells containing the vector described in claim 5.

7. The application of the coenzyme Q10 reductase mutant according to any one of claims 1-3, characterized in that: Used to prepare reduced coenzyme Q10.

8. The application according to claim 7, characterized in that: Using oxidized coenzyme Q10 and NADH as raw materials, and under the action of the coenzyme Q10 reductase mutant, formate dehydrogenase is used to catalyze the NADH recycling reaction to prepare reduced coenzyme Q10.

9. The application according to claim 8, characterized in that, The reaction parameters are as follows: reaction temperature 25-35℃, substrate oxidized coenzyme Q10 concentration 10-15mM, NADH concentration 2-5mM, ammonium formate concentration 20-30mM, sodium sulfite concentration 10-20mM; substrate solvent 0.1mol / L phosphate buffer, reaction pH 6.5-7.0, reaction time 2-6h, formate dehydrogenase activity 70-80U, coenzyme Q10 reductase mutant 500-600U, total reaction volume 100ml.

10. The use of the carrier of claim 5, or the engineered microorganism or cell of claim 6, in the preparation of coenzyme Q10 reductase, or reduced coenzyme Q10.

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