Ammonia dehydrogenase mutant and application of ammonia dehydrogenase mutant in preparation of milobalin chiral ketone intermediate DB01

Abstract

The invention belongs to the field of molecular biology and enzyme engineering, and relates to an ammonia dehydrogenase mutant and application thereof in preparation of a milobalin chiral ketone intermediate DB01, in particular to the ammonia dehydrogenase mutant, a recombinant host cell, a cell culture and a method for synthesizing the milobalin intermediate DB01 ((1R, 5S)-3-ethyl bicyclo [3.2. 0] hept-3-ene-6-ketone) through an enzymic method. The method provided by the invention solves the problems of high ketoalcohol separation cost, easy deterioration of the substrate in the separation process and the like in the existing industrial synthesis route, and has a wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and enzyme engineering, and relates to an ammonia dehydrogenase mutant and application thereof in preparing a mirogabalin chiral ketone intermediate DB01, in particular to an ammonia dehydrogenase mutant, a recombinant host cell, a cell culture, and a method for enzymatically synthesizing a mirogabalin intermediate DB01. BACKGROUND

[0002] Mirogabalin is developed by Japan's First Sanjiyao Corporation, and was approved for marketing by the Japan Pharmaceutical and Medical Device Integrated Agency on January 8, 2019. Mirogabalin, together with gabapentin and pregabalin, belongs to the drug category named gabapentin analogues, and is mainly used for treating peripheral neuropathic pain, including diabetic peripheral neuropathic pain, post-herpetic neuralgia and fibromyalgia, etc. Mirogabalin can preferentially and selectively bind to the α2δ-1 subunit of voltage-dependent calcium channels, but the efficacy is significantly higher than that of pregabalin. These calcium channels widely exist in the nervous system of various regions of the body to mediate pain transmission and processing, and mirogabalin has unique binding properties and long-acting effects. Its chemical structural formula is as follows:

[0003]

[0004] The current synthesis route is as follows: a chiral ketone is obtained by resolution, and after a plurality of reaction steps, the target product is obtained. The method for obtaining DB01 by resolution is mostly chemical resolution, and only one patent reports the use of carbonyl reductase for resolution, and then the product alcohol is derived using diacid. This patent is applied by the original research company of this drug (CN104245951B). After the resolution is completed, the separation of the ketone and the product alcohol in the reaction system using traditional ketone-alcohol separation methods is easy to cause the double bond of the substrate to isomerize, resulting in loss of raw materials, low yield of chiral ketone, and high cost of chiral ketone.

[0005]

[0006] The ketone substrate is resolved by ammonia dehydrogenase, and the ketone and the product amine can be separated by controlling the pH value of the aqueous phase. Therefore, it is necessary to develop an efficient biological catalytic method to efficiently realize the amination reaction of 3-ethylbicyclo[3.2.0]hept-3-en-6-one. SUMMARY

[0007] In previous research, the inventors screened a large number of ammonia dehydrogenases and identified an ammonia dehydrogenase derived from *Caldalkalibacillus thermomarum* capable of catalyzing the synthesis of (1S,5R)-3-ethylbicyclo[3.2.0]hept-3-en-6-amine from 3-ethylbicyclo[3.2.0]hept-3-en-6-one. To address the aforementioned problems in the prior art, this invention modifies the aforementioned ammonia dehydrogenase using various genetic engineering methods. By artificially introducing mutations, its catalytic performance is altered, resulting in ammonia dehydrogenase mutants. These mutants exhibit significantly enhanced activity in the synthesis of the chiral ketone intermediate DB01, enabling efficient synthesis of (1S,5R)-3-ethylbicyclo[3.2.0]hept-3-en-6-amine under mild reaction conditions, making them suitable for large-scale industrial production.

[0008] This invention constructs a mutant of wild-type ammonia dehydrogenase derived from Caldalkalibacillusthermarum, with an amino acid sequence as shown in SEQ ID NO:1, through site-directed mutagenesis. The mutant exhibits high enzyme activity, can tolerate high concentrations of substrate, and possesses good catalytic performance.

[0009] According to the literature ((a)Abrahamson, MJ; Vazquez-Figueroa, E.; Woodall, NB; Moore, JC; Bommarius, AS, Angew Chem Int Ed Engl 2012, 51(16), 3969-72; (b)Abrahamson, MJ; Wong, JW; Bommarius, AS, Advanced Synthesis & Catalysis 2013, 355(9), 1780-1786.), the carboxyl group of the amino acid dehydrogenase-binding amino acid residues lysine (K) and asparagine (N) are mutated to serine (S) and leucine (L), respectively. The K68 and N226 positions in SEQ ID NO:1 are mutated to obtain the amino acid dehydrogenase CtAmDH (CtAmDH K68S / N226L hereinafter referred to as WO).

[0010] In some embodiments, the ammonia dehydrogenase mutant of the present invention still exhibits high activity at high substrate concentrations (50-600 mM) and can be used to synthesize (1S,5R)-3-ethylbicyclo[3.2.0]hept-3-en-6-amine from 3-ethylbicyclo[3.2.0]hept-3-en-6-one, thereby retaining the chiral ketone (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one (DB01).

[0011] In some embodiments, the mutation sites of the ammonia dehydrogenase mutant of this disclosure include one or more substituted amino acids at positions 65, 68, 115, 266, 289, 293, and 296 of the sequence shown in SEQ ID NO.1. Specifically, the mutations are M65I; M65F; D115E; D115K; D115R; Y289S; A293L; L296A; L296F;

[0012] M65I and A293L; M65I and L296A or L296F; M65I and A293L and L296F.

[0013] In some embodiments, the recombinant polypeptide, isolated polynucleotide, nucleic acid construct, and recombinant expression vector of this disclosure respectively contain or express the above-mentioned ammonia dehydrogenase mutant, and can be applied to the industrial production of milobalin key intermediates.

[0014] This disclosure may include the preparation of milobalin key intermediates using the aforementioned ammonia dehydrogenase mutants, recombinant peptides, polynucleotides, nucleic acid constructs, recombinant expression vectors, recombinant host cells, and recombinant genetically engineered bacteria such as Escherichia coli.

[0015] In some specific embodiments, the ammonia dehydrogenase mutant can participate in the reaction in the form of pure enzyme, crude enzyme solution, host cells expressing it, cell fragments of host cells expressing it, or fermentation broth containing host cells expressing it. Host cells can be collected by centrifugation or filtration for host cell-catalyzed transformation.

[0016] In some preferred embodiments, the enzyme is involved in the reaction in the form of whole cells or crude enzyme solution.

[0017] In some specific steps of producing milobalin intermediates, 3-ethylbicyclo[3.2.0]hept-3-en-6-one is used as a substrate, and the concentration of the substrate is 50-800 mM, preferably 400-700 mM;

[0018] Some specific steps in the production of milobalin intermediates also include a coenzyme regeneration system, which is a system composed of glucose dehydrogenase and glucose, or a system composed of formate dehydrogenase and formate.

[0019] In some specific steps of producing milobalin intermediates, the pH of the reaction system can be selected from 7.0 to 9.0, preferably from 7.5 to 8.5, and more preferably from 8.0.

[0020] In some specific steps of producing key intermediates of milobaline, the reaction temperature can be selected as 20℃ to 40℃, preferably 30℃.

[0021] In some specific steps of producing key intermediates of milobaline, the reaction time can be selected to be more than 1 hour, preferably 12 to 28 hours, and more preferably 24 hours.

[0022] This invention involves directed evolution of the ammonia dehydrogenase AmDH (NCBI,WP_007505854.1) derived from Caldalkalibacillus thermomarum, resulting in an ammonia dehydrogenase mutant with enhanced enzyme activity and substrate tolerance. The chiral ketone (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one (DB01) was obtained through resolution. Compared to the wild type, the ammonia dehydrogenase mutant provided by this invention not only exhibits activity against simple ketone substrates but also shows significantly enhanced enzyme activity. In some embodiments, the ammonia dehydrogenase mutant of the present invention still exhibits high activity at high substrate concentrations. Therefore, the mutant of the present invention can synthesize (1S,5R)-3-ethylbicyclo[3.2.0]hept-3-en-6-amine from high concentrations (50-800 mM) of 3-ethylbicyclo[3.2.0]hept-3-en-6-one, thereby retaining the chiral ketone (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one (DB01).

[0023] The ammonia dehydrogenase mutant of the present invention has high catalytic efficiency, mild reaction conditions, short reaction time, and can be carried out at high substrate concentrations, thus saving costs for the synthesis of milobalin intermediate DB01.

[0024] Instruction manual illustrations

[0025] Figure 1 The gas phase spectrum of the racemic mixture of 3-ethylbicyclo[3.2.0]hept-3-en-6-one.

[0026] Figure 2 Gas phase spectra of (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one and chiral amine. Detailed Implementation

[0027] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0028] The amino dehydrogenase mutants constructed in this invention are all mutants of wild-type amino acid dehydrogenase (NCBI,WP_007505854.1) derived from Caldalkalibacillus thermoma.

[0029] Example 1: Construction of a library of W0 mutants of ammonia dehydrogenase and screening of mutants.

[0030] 1. The first round of revitalization transformation

[0031] The simulated protein structure of ClAmDH was obtained through Alphafold2 modeling, and its substrate binding pocket was selected. The amino acid residues within the range (respectively, residues 40, 41, 42, 43, 44, 45, 46, 48, 57, 58, 60, 61, 62, 64, 65, 69, 72, 79, 80, 81, 82, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 123, 134, 135, 146, 147, 149, 150, 1) Saturation mutations were performed at positions 51, 154, 183, 184, 209, 242, 243, 244, 245, 263, 264, 265, 267, 268, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, and 300. Mutation primers were designed using the degenerate codon NNK, with pET28a-W0 as the template. A two-step PCR method was used, employing the high-fidelity polymerase FastPfu-DNA.

[0032] 1) Construction of pET28a-ClAmDH plasmid

[0033] The amino acid sequence of the wild-type amino acid dehydrogenase is shown in SEQ ID NO:1. The corresponding nucleotide sequence of the W0 mutant based on the wild-type was fully synthesized and cloned into the restriction endonuclease sites NdeI and EcoRI of the pET-28a vector to obtain the recombinant plasmid pET-28a-W0. This plasmid was further transformed into the expression host E. coli BL21(DE3), and positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET-28a-W0.

[0034] 2) Construction of a single-point mutant library of ammonia dehydrogenase

[0035] Using pET28a-W0 as a template, amplification was performed using the primers in Table 1. The PCR reaction system and reaction conditions are as follows:

[0036] A. Step PCR reaction system and reaction conditions

[0037] Round 1: In a 25 μL PCR reaction system, add 15 ng template, 5 μL 10× buffer, 2 L dNTPs (2.5 mM), 0.5 μL each of a pair of mutant primers (10 μM), and 0.5 μL FastPfu DNA polymerase. Add sterile distilled water to a final volume of 25 μL. The first step PCR reaction program is as follows: ① 95℃ pre-denaturation for 2 min, ② 95℃ denaturation for 20 sec, ③ Tm-5℃ annealing for 20 sec, ④ 72℃ extension for 15 sec, ⑤ 72℃ final extension for 5 min. Perform steps ② through ④ for a total of 30 cycles.

[0038] Step 2: PCR reaction system and reaction conditions

[0039] Round 2: To a 50 μL PCR reaction system, add 30 ng of template, 10 μL of 5×FastPfu DNA polymerase buffer, 5 μL of dNTP (2 mM), 1 μL each of a pair of mutant primers, 1 μL of Pfu polymerase, 2 μL of MgSO4 (25 mM), and distilled water to a final volume of 50 μL. The second step of the PCR reaction program is as follows: ① 95℃ pre-denaturation for 2 min, ② 95℃ denaturation for 20 sec, ③ 60℃ annealing for 45 sec, ④ 72℃ extension for 3 min, ⑤ 72℃ final extension for 5 min. Perform steps ② through ④ for a total of 30 cycles.

[0040] After verifying the PCR products obtained in the above steps by agarose gel electrophoresis, the restriction endonuclease DpnI was added and digested at 37°C for 2 h. The digested products were transformed into E. coli BL21(DE3) competent cells and plated on plates containing 50 μg / mL kanamycin antibiotic. The plates were then incubated statically at 37°C for approximately 12 h, resulting in single colonies and the ammonia dehydrogenase mutant library. Simultaneously, the pET28a-W0 plasmid was transformed into E. coli BL21(DE3) competent cells and plated on plates containing 50 μg / mL kanamycin antibiotic. The plates were then incubated statically at 37°C for approximately 12 h, resulting in single colonies and the strain expressing the ammonia dehydrogenase mutant.

[0041] Table 1. Primer sequences from the first round of modification

[0042]

[0043]

[0044] B. Induced expression of ammonia dehydrogenase mutants

[0045] The monoclonal colonies obtained after culturing in step 2 above were picked and transferred to 4 mL of LB liquid medium containing kanamycin (50 μg / L) (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl), and cultured overnight at 37°C and 200 rpm to obtain the culture medium. The culture medium was then inoculated into fermentation medium (LB liquid medium) at a 1% (v / v) inoculation rate and cultured on a shaker at 37°C and 200 rpm until OD... 600 The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The mixture was induced at 25°C and 200 rpm for 8-12 hours. The bacterial cells were collected by centrifugation at 6000 g, and then subjected to high-pressure disruption to obtain a crude enzyme solution of ammonia dehydrogenase for subsequent enzyme activity assays.

[0046] C. Screening for ammonia dehydrogenase mutants

[0047] The screening method involved detecting the decrease in NADPH at 340 nm. The specific reaction method was as follows: 5 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 20% (v / v) dimethyl sulfoxide (DMSO), 0.25 mg / mL NADPH, 80 μL crude enzyme solution, and 100 mM sodium phosphate buffer (pH 7.5) to a final volume of 200 μL. NADPH exhibits characteristic absorbance at 340 nm. The decrease in absorbance at 340 nm was detected using a microplate reader; if the enzyme activity was relatively high, NADPH consumption was faster, and the slope of the decrease curve was steeper. Beneficial mutation sites that improved enzyme activity were identified through screening: 65, 115, 289, 293, and 296, specifically mutants 1–8.

[0048] 2. Second round of revitalization transformation

[0049] Based on the results of saturation mutation, using mutant 1 as a template, saturation mutations were performed at points 115 and 289, respectively, but no mutants improved compared to mutants 5 and 6 were obtained. Using mutant 1 as a template again, saturation mutation was performed at point 293, resulting in mutant 10: M65I / A293L. Using mutant 10 as a template, saturation mutation was performed at point 296, resulting in mutant 11: M65I / A293L / L296AF.

[0050] Table 3. Relative activity of W0 and its mutants to substrates

[0051] Mutant M65 D115 Y289 A293 L296 Relative activity W0 1.0 Mutant 1 I 5.0 Mutant 2 F 4.1 Mutant 3 K 6.4 Mutant 4 R 8.1 Mutant 5 E 12.1 Mutant 6 S 7.2 Mutant 7 L 5.3 Mutant 8 A 9.1 Mutant 9 F 6.1 Mutant 10 I L 21.6 Mutant 11 I L F 31.6

[0052] Example 2: Synthesis of milobarin intermediate using crude enzyme solution of mutant 11 as catalyst.

[0053] Mutant 11 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). The crude enzyme solution after whole-cell lysis (20 mg / mL) was used as the biocatalyst, along with 100 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 1 M ammonium formate, and 0.5 g / L NADP. + 3 U / mL formate dehydrogenase and sodium phosphate buffer (pH 8.0, 100 mM) were added, and the reaction was carried out at 25 °C for 14 hours. After the reaction, the pH was adjusted to below 5.0 with hydrochloric acid, and the mixture was extracted three times with an equal volume of methyl tert-butyl ether. The organic phases were combined and dried over anhydrous sodium sulfate to obtain (1R,5S)-3-ethylbicyclo[3.2.0]hepten-3-en-6-one (DB01). After removing the solvent under reduced pressure, the yield was 42% and the ee value was 99%. Figure 1 and Figure 2 The pH was adjusted to above 10 with sodium hydroxide, and the aqueous phase was extracted with dichloromethane. The organic phases were combined, dried, and the chiral amine was collected by rotary evaporation. The ee value was 99%, and the separation yield was 43%. The reaction progress was monitored by gas chromatography. The gas chromatography conditions were: helium as carrier gas, CP-ChiraSil-DEX CB column (25m×0.25mm×0.25mm), and the temperature program was: 80℃ (2min) – 10℃ (min). -1 -180℃ (3 min).

[0054] Example 3: Whole-cell catalytic synthesis of milobarin intermediate using mutant 11

[0055] Mutant 11 was obtained by inducing expression according to the method of Example 1. The bacterial cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. Other conditions were the same as in Example 2. The yield of chiral ketones was 43% with an ee value of 99%; the yield of chiral amines was 43% with an ee value of 99%.

[0056] Example 4: Synthesis of milobalin intermediate using crude enzyme solution of mutant 5.

[0057] Mutant 5 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). The crude enzyme solution after lysis (30 mg / mL) was used as the biocatalyst, and other conditions were the same as in Example 2. The yield of chiral ketones was 40%, with an ee value of 99%; the yield of chiral amines was 42%, with an ee value of 99%.

[0058] Example 5: Synthesis of milobarin intermediate using crude enzyme solution of mutant 11 as catalyst.

[0059] Mutant 11 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). The crude enzyme solution after whole-cell lysis (40 mg / mL) was used as the biocatalyst. The reaction system contained 400 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 2 M ammonium formate, and 0.5 g / L NADP. + The mixture was prepared with 7 U / mL formate dehydrogenase and sodium phosphate buffer (pH 8.0, 100 mM), and reacted at 30°C for 24 hours. Post-treatment conditions were the same as in Example 2. The yield of chiral ketones was 45%, with an ee value of 99%; the yield of chiral amines was 43%, with an ee value of 99%.

[0060] Example 6: Synthesis of milobarin intermediate using crude enzyme solution of mutant 11 as catalyst.

[0061] Mutant 11 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). The crude enzyme solution after whole-cell lysis (60 mg / mL) was used as the biocatalyst. The reaction system contained 700 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 5 M ammonium formate, and 0.5 g / L NADP. + The mixture was prepared with 10 U / mL formate dehydrogenase and sodium phosphate buffer (pH 8.0, 100 mM), and reacted at 30°C for 30 hours. Post-treatment conditions were the same as in Example 2. The yield of chiral ketones was 45%, with an ee value of 99%; the yield of chiral amines was 43%, with an ee value of 99%.

[0062] Example 7: Synthesis of milobarin intermediate using crude enzyme solution of mutant 11 as catalyst.

[0063] Mutant 11 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). The crude enzyme solution after whole-cell lysis (100 mg / mL) was used as the biocatalyst. The reaction system contained 800 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 5 M ammonium formate, and 0.5 g / L NADP. + The mixture was prepared with 14 U / mL formate dehydrogenase and sodium phosphate buffer (pH 8.0, 100 mM), and reacted at 30°C for 36 hours. Post-treatment conditions were the same as in Example 2. The yield of chiral ketones was 46%, with an ee value of 99%; the yield of chiral amines was 44%, with an ee value of 99%.

[0064] Example 8: Synthesis of milobalin intermediate using whole-cell catalysis of mutant 11

[0065] Mutant 11 was obtained by inducing expression according to the method in Example 1. The bacterial cells were collected by centrifugation (6000 rpm). Using 100 mg / mL whole cells as a biocatalyst, the reaction system contained 500 mM 3-ethylbicyclo[3.2.0]hepta-3-en-6-one, 5 M ammonium formate, and 0.5 g / L NADP. + The mixture was prepared with 10 U / mL formate dehydrogenase and sodium phosphate buffer (pH 8.0, 100 mM), and reacted at 30°C for 24 hours. Post-treatment conditions were the same as in Example 2. The yield of chiral ketones was 46%, with an ee value of 99%; the yield of chiral amines was 44%, with an ee value of 99%.

Claims

1. An ammonia dehydrogenase mutant, characterized in that, It is an amino dehydrogenase mutant with mutations at one or more sites, including sites 65, 115, 289, 293 and 296, corresponding to the amino acid sequence shown in SEQ ID NO.

1.

2. The ammonia dehydrogenase mutant as described in claim 1, characterized in that, The mutations are M65I; M65F; D115E; D115K; D115R; Y289S; A293L; L296A; L296F; M65I and A293L; M65I and L296A or L296F; M65I and A293L and L296F; Preferably, the amino acid sequence corresponding to SEQ ID NO. 1 contains one of the following mutations: D115E, or M65I and A293L and L296F.

3. The encoding gene of the ammonia dehydrogenase mutant as described in claim 1 or 2.

4. An expression vector containing the encoding gene of the ammonia dehydrogenase mutant as described in claim 1 or 2.

5. Recombinant cells containing the coding gene of the ammonia dehydrogenase mutant as described in claim 1 or 2, specifically such as Escherichia coli.

6. The ammonia dehydrogenase mutant as described in claim 1 or 2, or the gene encoding the ammonia dehydrogenase mutant as described in claim 3, in the preparation (1 R 5 S Application in 3-ethylbicyclo[3.2.0]hepten-3-en-6-one.

7. A method for preparing (1 R 5 S The method for 3-ethylbicyclo[3.2.0]hept-3-en-6-one, characterized in that, Using an ammonia dehydrogenase mutant as described in claim 1 or 2 (the ammonia dehydrogenase mutant is in the form of pure enzyme, crude enzyme solution, host cells expressing it, cell fragments of host cells expressing it, or fermentation broth containing host cells expressing it) as a catalyst, and 3-ethylbicyclo[3.2.0]hept-3-en-6-one as a substrate, a catalytic reaction is performed to resolve (1) R 5 S )-3-ethylbicyclo[3.2.0]hept-3-en-6-one.

8. The method as described in claim 7, characterized in that, Using wet bacterial cells obtained by fermentation culture of engineered bacteria expressing the coding gene of the ammonia dehydrogenase mutant as described in claim 3 as a catalyst, 3-ethylbicyclo[3.2.0]hept-3-en-6-one and inorganic ammonium salt as substrates, a buffer solution with a pH of 7.0-11.0 as the reaction medium, and an additional coenzyme regeneration system, the catalytic reaction was carried out at 25℃-50℃.

9. The method as described in claim 7 or 8, characterized in that, In the catalytic reaction, the concentration of the catalytic substrate in the reaction system is 50-800 mM, more preferably 400-700 mM; the amount of bacteria in the reaction system is 10-150 g / L, more preferably 30-70 g / L; the pH of the reaction system is 7.0-9.0, preferably 8.0; and the reaction temperature is 25 ℃-50 ℃, preferably 30 ℃.

10. The method as described in claim 9, characterized in that, The inorganic ammonium salt added to the reaction mixture is ammonium formate, ammonium chloride, or ammonia, and the reaction is carried out at 150 rpm - 250 rpm for 5-25 hours. The coenzyme regeneration system is either glucose dehydrogenase / glucose system or formate dehydrogenase / formate system.

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