A method for preparing monoamine oxidase and boceprevir bicyclic proline fragment

Abstract

The invention discloses a method for preparing a monoamine oxidase and a bicyclic proline fragment of boceprevir. By mutating the monoamine oxidase sequence, the interaction between amino acid residues in the local area of ​​the enzyme molecule and between the enzyme and the environment is changed, and a monoamine oxidase engineered bacterium with higher conversion efficiency is obtained. The synergistic effect of monoamine oxidase and catalase is utilized to reduce substrate residues and realize industrial production. In the preparation process, sodium pyrosulfite is added to the reaction system in a flow addition manner together with the substrate, so as to reduce the influence of the substrate on the enzyme activity and improve the conversion rate of the enzyme.

Description

Technical Field

[0001] The present invention relates to the field of biological enzyme catalysis, and in particular to a method for preparing monoamine oxidase and a bicyclic proline fragment of boceprevir. Background Art

[0002] The Chinese name of Boceprevir is: (1R,2S,5S)-N-(4-amino-1-cyclobutyl-3,4-dioxobutane-2-yl)-3-[(2S)-2-(tert-butylcarbamoylamino)-3,3-dimethylbutanoyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide. 6,6-dimethyl-3-azabicyclo[3.1.0]hexane derivatives are important intermediates for the synthesis of Boceprevir. Boceprevir can truncate and modify ketoamide amino acid residues, greatly inhibiting the replication of hepatitis C virus RNA, not only improving the cure rate but also shortening the treatment time. Its effect is better than that of pegylated interferon α or ribavirin, bringing good news to the majority of hepatitis C patients.

[0003] (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide ( Figure 1 The part shown in A in the middle is the bicyclic proline fragment of boceprevir, which is an important intermediate in the synthesis of boceprevir. Currently, the main methods for the bicyclic proline fragment of boceprevir are chemical method and biological enzyme catalysis.

[0004] The chemical method uses 6,6-dimethyl-3-azabicyclo[3.1.0]hexane hydrochloride as the raw material, and after amino protection, uses 4,4'-difluorobenzophenone as a chiral inducing agent. Under the action of a hydrogen extraction reagent, it reacts with 1,2,3,4-tetrahydro-1-naphthylamine at 30-35°C for 3-4 hours. Finally, the amino protecting group is removed and an acid is added to form a salt to obtain the bicyclic proline fragment of boceprevir. However, a low temperature of -78°C is required during the reaction process. The reaction conditions are too harsh and the equipment requirements are relatively high, resulting in increased production costs. Secondly, sec-butyl lithium is highly flammable and immediately releases highly flammable gas when it comes into contact with water. It can spontaneously combust in the air. This determines that the reaction requires anhydrous and oxygen-free conditions, and great caution is required during operation.

[0005] Compared with chemical methods, biocatalysis is efficient, environmentally friendly, and has mild reaction conditions. However, the conversion rate of monoamine oxidase is low and the substrate residue is large during the conversion process, which restricts industrial production. Changes in the type and number of forces within protein molecules will have an important impact on the catalytic substrate efficiency and substrate residue of enzyme molecules. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a monoamine oxidase for biocatalytic synthesis of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-sulfonate; another purpose of the present invention is to provide a nucleotide sequence encoding the aforementioned monoamine oxidase, a recombinant expression vector and a monoamine oxidase engineered bacterium; another purpose of the present invention is to provide a conversion enzyme solution containing both monoamine oxidase and catalase; another purpose of the present invention is to provide a method for biocatalytic synthesis of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo [3.1.0] hexane-2-sulfonate.

[0007] Technical solution: The monoamine oxidase described in the present invention is mutated at least one of positions 65, 120, 147, 176, 213, 252 and 317 based on the amino acid sequence shown in SEQ ID No.1. SEQ ID No.1 is derived from the genome template of Aspergillus niger CBS 513.88 strain, and a gene fragment of monoamine oxidase MAO is obtained by amplification by designing primer pairs. Preferably, the mutation sites are at positions 65, 147, 176, 213, 252 and 317.

[0008] The amino acid mutation types of the mutation sites include: T65G or T65V; N120D; E147A; Y176H, Y176S or Y176A; L213F or L213M; D252T, D252G, D252S or D252A; Q317H, Q317C or Q317W. Preferably, the threonine Thr at the 65th amino acid is mutated to valine Val; the glutamic acid Glu at the 147th amino acid is mutated to alanine Ala; the tyrosine Tyr at the 176th amino acid is mutated to histidine His or alanine Ala; the leucine Leu at the 213th amino acid is mutated to phenylalanine Phe or methionine Met; the aspartic acid Asp at the 252th amino acid is mutated to glycine Gly, serine Ser or alanine Ala; the glutamine Gln at the 317th amino acid is mutated to histidine His, cysteine ​​Cys or tryptophan Trp.

[0009] Preferably, at least one of the following mutation types is included: Glu at position 147 is mutated to Ala; Tyr at position 176 is mutated to His or Ala; Leu at position 213 is mutated to Phe; Aspartic acid Asp at position 252 is mutated to Gly; Glutamine Gln at position 317 is mutated to His, Cysteine ​​Cys or Tryptophan Trp. These amino acid sequences are shown in SEQ ID No. 2-19.

[0010] Most preferably, the aspartic acid Asp at position 252 is mutated to glycine Gly, and the glutamine Gln at position 317 is mutated to histidine His or tryptophan Trp.

[0011] The present invention provides a recombinant expression vector based on the aforementioned monoamine oxidase, comprising a nucleotide sequence encoding any one of the amino acid sequences shown in SEQ ID No. 2-19. The recombinant expression vector is selected from any one of pET, pGEX, and pMAL as a vector, preferably pET-28(a) plasmid.

[0012] The constructed monoamine oxidase recombinant expression vector is transformed into a host cell to obtain a recombinant monoamine oxidase engineered bacterium. The host cell is selected from any one including but not limited to Escherichia coli, Bacillus glutamicum, and Bacillus subtilis, preferably Escherichia coli BL21 (DE3).

[0013] The same method as above was used to further construct a recombinant catalase engineered bacterium. The catalase was derived from Bacillus pumilus ML 413 strain (GenBank: KT963080.1), and its nucleotide sequence was shown in SEQ ID No. 20. The fragment was inserted into an expression plasmid to obtain a recombinant catalase expression vector; and then the vector was transferred into a host cell of Escherichia coli to obtain a recombinant catalase engineered bacterium.

[0014] The present invention provides a converting enzyme solution, which is obtained by fermenting, culturing, cell breaking and flocculating the monoamine oxidase engineering bacteria and the catalase engineering bacteria, collecting the supernatants and mixing them in proportion. The mixing ratio of the monoamine oxidase supernatant and the catalase supernatant is 5-50:1, preferably 10-30:1 to obtain synergistic effect, and the best synergistic effect can be obtained when the ratio reaches 20:1.

[0015] The flocculation treatment is performed by using any one of polyethyleneimine, chitosan, and polyacrylamide as a flocculant; preferably, 0.12-0.18 w / v% polyethyleneimine is used.

[0016] The present invention provides a method for biocatalytically synthesizing sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate (a compound shown in Formula II), which is to use 6,6-dimethyl-3-azabicyclo[3.1.0]hexane (a compound shown in Formula I) as a substrate, oxygen as an oxidant, and use the invertase liquid to perform a desymmetric reaction, and simultaneously perform an addition reaction with sodium pyrosulfite to generate a corresponding aminosulfonate. The catalytic process route is as follows:

[0017]

[0018] The enzyme-catalyzed reaction product (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate sodium is reacted with a cyaniding agent in cyclopentyl methyl ether (CPME), followed by a Pinner reaction and extraction with methyl tert-butyl ether (MTBE). The product (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxylic acid methyl ester hydrochloride is finally crystallized in the form of hydrochloride, which is an important intermediate for the synthesis of boceprevir.

[0019] In the biocatalytic process of the present invention, the control of substrate concentration is particularly important. The concentration of the substrate 6,6-dimethyl-3-azabicyclo[3.1.0]hexane is the main factor determining the enzyme catalytic reaction rate. When the substrate concentration is too high, the enzyme reaction rate will decrease due to substrate inhibition; when the substrate concentration is very low, the enzyme is not completely saturated with the substrate, and the catalytic reaction efficiency is affected. Therefore, it is preferred to control the substrate concentration in the reaction system by configuring the substrate solution and using a peristaltic pump feed method.

[0020] The present invention adopts a bubbling reactor to continuously and uninterruptedly provide oxygen, wherein the oxygen content of the gas is ≥21%. The substrate is added to the reaction system in a flow addition manner, and the substrate concentration in the reaction system is controlled at 5-10 g / L.

[0021] Furthermore, the amount of the invertase solution added is 20-25 v / v% of the total reaction system. The amount of the invertase solution added includes the amount of enzyme supplemented in the feed reaction system.

[0022] Furthermore, the pH of the enzyme catalytic reaction is controlled at 7.0-7.8, preferably 7.4. The catalytic reaction time is 36-48h, preferably 42h, the reaction temperature is 25±3°C, and the substrate residue is monitored during the reaction. The substrate residue after 42h reaction measured for the single-site mutant of the above-mentioned monoamine oxidase does not exceed 3.50%; the double-site mutation D252G+Q317H is particularly preferred, and the substrate residue after 42h reaction reaches 0%; similarly, the substrate residue of D252G+Q317W reaches 0.03%, and the single-site mutant is most preferably Q317H, with a substrate residue of 0.06%.

[0023] The sodium pyrosulfite and the substrate are prepared into a substrate solution according to a molar mass ratio of 1:1, and added into the reaction system in a flow-feed manner together with the substrate.

[0024] Beneficial effects:

[0025] 1. The present invention obtains a monoamine oxidase engineered bacterium with higher conversion efficiency by mutating the monoamine oxidase sequence and changing the interaction between amino acid residues in the local area of ​​the enzyme molecule and between the enzyme and the environment. In particular, the conversion rate of the D252G and Q317H site mutant strains is increased by 4.2 times compared with the original strain, the substrate residue is reduced, and the target product obtained by the enzyme desymmetric reaction of the substrate has a better enantioselectivity of ≥99% ee, thereby realizing industrial production.

[0026] 2. By further optimizing the technical means of combining monoamine oxidase and catalase, a solution was obtained that achieved the best synergistic effect at a ratio of 20:1.

[0027] 3. In the process of biocatalytic synthesis of boceprevir bicyclic proline fragment, sodium pyrosulfite is added to the reaction system together with the substrate in a flow-feed manner, which reduces the effect of the substrate on the enzyme activity and improves the conversion efficiency of the enzyme in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the chemical structural formula of boceprevir, wherein A represents a bicyclic proline fragment;

[0029] Figure 2 This is a gas chromatogram of the substrate residue after 42 hours of conversion of the production process of Example 2. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] Example 1 Preparation of monoamine oxidase and catalase conversion enzyme solution

[0032] 1. Construction of monoamine oxidase cloning strain

[0033] After Aspergillus niger CBS 513.88 (GenBank: XP_001395406.1) was resuscitated and subcultured, the genome was extracted as a DNA template, and primers F / R were designed according to the sequence of the gene encoding monoamine oxidase protein (MAO), forward primer (MAO-F-BamH I): 5'-CGCGGATCCATGACCAGCCGCGACGGCTA-3'; reverse primer (MAO-R-Xho I): 5'-CCGCTCGAGCAGGCGGGCCTTCACCTCG-3'.

[0034] PCR reaction system: 10×Ex Taq Buffer 2.5μL, dNTP (2.5mmol / L) 1μL, upstream and downstream primers (10μmol / L) 0.5μL each, template DNA (50ng / μL) 1μL, Ex Taq enzyme (5U / μL) 0.25μL, ddH2O 19.25μL, total volume 25μL.

[0035] PCR reaction conditions: pre-denaturation at 94°C for 1 min, enter the cycle: denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 1 min, enter 30 cycles, and finally extend at 72°C for 5 min.

[0036] After PCR, gel verification and product recovery were performed. The target fragment was recovered by electrophoresis detection and gel cutting. The recovered target protein was double-digested with BamH I-Xho I and connected with the pET-28a plasmid that was also double-digested with BamH I-Xho I at 16°C overnight. After connection, the expression vector pET-28a-MAO encoding the MAO gene was obtained.

[0037] 2. Construction of monoamine oxidase expression strain

[0038] The expression vector pET-28a-MAO encoding the MAO gene was heat-shock transformed into competent E. coli BL21 (DE3), and positive transformants were screened on kanamycin-resistant plates, and PCR and double enzyme digestion verification were performed. After successful PCR verification, the recombinant plasmid was extracted, and the extracted plasmid was digested and sequenced respectively to verify that a recombinant monoamine oxidase clone strain was obtained, and its amino acid sequence is shown in SEQ ID NO.1.

[0039] 3. Site-directed mutagenesis

[0040] The three-dimensional structure of monoamine oxidase was automatically modeled using SWISS-MODEL, and then the model was displayed using PyMOL software to analyze its structural characteristics. The homologous sequences of monoamine oxidase amino acids from different sources were compared using MEGA-X software to analyze the conserved amino acid sequence of the enzyme. The interactions between amino acid residues were analyzed using PyMOL software. The molecular docking of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane and MAO was performed using Discovery Studio software, and the amino acid sites at positions 65, 120, 147, 176, 213, 252, and 317 were selected for site-directed saturation mutagenesis.

[0041] Degenerate primers were designed according to the selected loci as follows:

[0042]

[0043] After PCR, 10 μL of the PCR products were respectively taken out and mixed, and then 10 μL of restriction endonuclease DpnI was added to remove the template plasmid. After recovery by DNA gel recovery kit, heat shock transformation was carried out into Escherichia coli BL21 (DE3) competent cells, and positive transformants were screened on kanamycin resistance plates.

[0044] 4. Construction of catalase expression strain

[0045] The nucleotide sequence of catalase derived from Bacillus pumilus ML 413 (GenBank: KT963080.1) was shown in SEQ ID No. 20. Shanghai Jierui Biotechnology Co., Ltd. was commissioned to synthesize the gene containing the vector pET-28a(+), and heat-shock transformed into Escherichia coli BL21(DE3) competent cells. Positive transformants were screened on kanamycin resistance plates, and PCR and double enzyme digestion verification were performed.

[0046] 5. Preparation of Monoamine Oxidase and Catalase Enzyme Solutions

[0047] The monoamine oxidase engineered bacteria and catalase engineered bacteria obtained after the above-mentioned site-directed mutagenesis were each inoculated onto a slant containing kanamycin resistance. After culturing at 37°C for 16 h, a loop was picked up from the slant with an inoculation loop and transferred to a TB medium containing kanamycin resistance. The culture was shaken at 37°C and 200 rpm until OD600 = 0.6. The culture was inoculated into a fermentation medium containing kanamycin resistance at an inoculum concentration of 1% by volume. The culture was shaken at 37°C and 200 rpm until OD600 = 0.5. IPTG was then added at a final concentration of 0.3 mM and cultured at 24°C and 200 rpm for 16 h. The fermentation broth was centrifuged at 4°C and 5000 rpm for 15 min, and the supernatant was discarded to collect the wet cells. The wet bacteria were added into distilled water at a concentration of 300 g / L, and ultrasonically disrupted at 400 W in an ice bath, with an interval of 3 s for each disruption for 5 s to obtain a cell suspension. After adding polyethyleneimine at a final concentration of 0.15 w / v% to the cell suspension for flocculation, the suspension was centrifuged at 4°C and 5000 rpm for 15 min. The supernatant obtained was mixed at a volume ratio of 20:1 to obtain the invertase solution.

[0048] 6. MAO single point mutant transformation assay substrate residue

[0049] Reaction system: Add 150 μL 0.1M PBS (pH = 7.4) buffer to each well of a 96-well plate, 50 μL of the above-mentioned invertase solution, 10.0 mg 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, 11.5 mg sodium pyrosulfite, and react at 25°C overnight. After the reaction, gas chromatography was used to analyze the substrate residue. The mutation point with a substrate residue lower than that of the initial strain WT was selected, and the results are shown in Table 1.

[0050] Table 1 Mutants with higher transformation rates than the initial strain

[0051] Mutation site Amino acid sequence Substrate residue (%) Conversion rate (%) WT SEQ ID No.1 3.84 23.2 T65G SEQ ID No.2 2.22 55.6 T65V SEQ ID No.3 3.04 39.2 N120D SEQ ID No.4 2.62 47.6 E147A SEQ ID No.5 1.45 71.0 Y176H SEQ ID No.6 0.96 80.8 Y176A SEQ ID No.7 1.52 69.6 Y176S SEQ ID No.8 3.59 28.2 L213F SEQ ID No.9 0.25 95.0 L213M SEQ ID No.10 2.38 52.4 D252G SEQ ID No.11 0.11 97.8 D252A SEQ ID No.12 1.97 60.6 D252S SEQ ID No.13 2.39 52.2 D252T SEQ ID No.14 3.23 35.4 Q317H SEQ ID No.15 0.06 98.8 Q317W SEQ ID No.16 0.19 96.2 Q317C SEQ ID No.17 0.87 82.6 D252G and Q317H SEQ ID No.18 0 100 D252G and Q317W SEQ ID No.19 0.03 99.4

[0052] 7. Scale-up and Preparation of Invertase Solution

[0053] The monoamine oxidase engineering bacteria with double mutations (D252G and Q317H) of MAO-18 were selected and cultured with the catalase engineering bacteria by high-density method.

[0054] Inoculate 100mL of pre-cultured monoamine oxidase engineering bacteria and catalase engineering bacteria seed liquid into two 7L fermentation tanks with 5L sterilized culture medium, start stirring, the stirring speed is 500rpm at the beginning, and increase to 700rpm after 3h; at the same time, control the ventilation volume: sterile air is introduced at 1vvm (4L / min) at the beginning, and increases to 1.2vvm (6L / min) after 3h. During the cultivation process, the dissolved oxygen drops rapidly after 2h of fermentation and rises rapidly after about 5h. At this time, feed is started to control the dissolved oxygen at about 30%. The pH will rise slowly in the early stage of fermentation, and the pH will drop after feeding. After that, the pH can be controlled between 6.3-6.8 with dilute ammonia water. The temperature is controlled at 37℃ in the early stage, and the temperature is slowly reduced to 30℃ 1-2h after the start of feeding. During the fermentation process, the OD value is monitored by a UV spectrophotometer. When OD600 rises to 15-25, 0.3mM inducer IPTG is added, and then the temperature is lowered and the induction is continued for 14-16h. When the induction is completed, the OD600 is about 60 when the tank is removed, and the fermentation liquid is obtained. The fermentation liquid is centrifuged at 4°C and 5000rpm for 15min. After the centrifugation, the supernatant is discarded and the wet cells are collected. The wet cells are added to distilled water at a concentration of 300g / L, and 10g / L of potassium dihydrogen phosphate is added to assist dissolution. The bacterial liquid is resuspended, and an ice bath is used. Ultrasonic disruption is performed at 400w. Every 5s of disruption is separated by 3s to obtain a cell disruption suspension. After adding a final concentration of 0.15w / v% polyethyleneimine to the cell suspension for flocculation, it is centrifuged at 4°C and 5000rpm for 15min. The resulting supernatant is mixed at a volume ratio of 20:1 to obtain the invertase solution.

[0055] Example 2

[0056] This example describes in detail the process of biocatalytic synthesis of sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate in a 350 mL reaction system.

[0057] Preparation of substrate solution: Add 22.87 g substrate (6,6-dimethyl-3-azabicyclo[3.1.0]hexane) and 144.38 g purified water into a container, stir for 15±5 minutes, then add 26.25 g sodium metabisulfite and stir until the solid is completely dissolved and the liquid is uniform without stratification.

[0058] Reaction system: Add 100g of purified water to the reactor, heat to 25±3℃, start stirring, add 1.17g of raw material, 1.28g of sodium pyrosulfite and stir for 10min, let in air, adjust the pH to 7.3, add 0.1g of defoamer, and after confirming the temperature and pH, add 40mL of the above-mentioned invertase solution to start the reaction. During the reaction, maintain the pH at 7.4 with a 2M NaOH aqueous solution until the reaction is completed. Add the substrate solution at the beginning of the reaction, and control the flow rate at 0.75mL / 10min.

[0059] The first enzyme supplementation time is about 9 hours, adding 0.8g sodium pyrosulfite and 20mL of the above-mentioned invertase solution. The second enzyme supplementation time is about 27 hours, which is determined according to the reaction situation, adding 0.8g sodium pyrosulfite and 20mL of the above-mentioned invertase solution. The substrate residues were detected at different sampling times, and the results are shown in Table 2:

[0060] Table 2 Detection of substrate residues at different sampling times

[0061] Time Substrate Residue % 5 0.19 21 0.06 27 0.05 42 0

[0062] Figure 2 This is the gas chromatogram after 42 hours of reaction. It can be seen from the figure that substrate peak A is not seen at this time, indicating that the substrate has been completely reacted and the residual amount is 0%.

[0063] Test Example 3

[0064] This test example explores the effect of the amount of invertase solution added on the conversion rate for the preparation process of Example 2. The amount of invertase solution added was adjusted, and other conditions were the same. The results of sampling and testing the substrate residue and conversion rate after 42 hours of reaction are shown in Table 3.

[0065] Table 3 Effect of different enzyme solution addition amounts on conversion rate

[0066] Comparative Example 1 Comparative Example 2 Comparative Example 3 Enzyme solution addition amount (mL) 60 70 80 Substrate residue (%) 1.98 0.69 0 Conversion rate (%) 71.2％ 89.9 100

[0067] It can be seen that the conversion rate can be significantly improved and the substrate residue can be reduced when the amount of invertase solution added exceeds 20% of the total reaction volume, preferably between 20-25 v / v%.

[0068] Test Example 4

[0069] This experiment explores the effect of different flow rates on conversion rate for the preparation process of Example 2. The substrate concentration in the reaction system is controlled by adjusting the flow rate of the feed reaction. After the feed of the substrate solution is completed, samples are taken to detect the substrate residue and conversion rate. The results are shown in Table 4.

[0070] Table 4 Effect of different flow rates on conversion

[0071] Test Example 1 Test Example 2 Test Example 3 Flow rate (mL / 10min) 0.50 0.75 1.00 Substrate flow time (h) 60 40 30 Substrate residue (%) 0 0.03 2.47 Conversion rate (%) 100 99.6 64.0

[0072] It should be noted that the substrate flow rate will affect the reaction time. In this test example, the substrate residue was 0.03% at 40h at 0.75mL / 10min. When the reaction time was extended to 42h, the substrate residue was 0%. At a flow rate of 0.50mL / 10min, it took 60h to add all the substrates into the reaction system, and the reaction time would be extended accordingly. It can be seen that too high a flow rate will affect the substrate conversion efficiency, while too low a flow rate will affect the production efficiency. A flow rate of 0.75mL / 10min can take both into account at the same time.

[0073] Example 5

[0074] This example provides a method for biocatalytic synthesis of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate in a 7 L reaction system (10 L reactor).

[0075] Preparation of substrate solution: Add 457.5 g substrate (6,6-dimethyl-3-azabicyclo[3.1.0]hexane) and 2888 g purified water into a container, stir for 15±5 minutes, then add 525 g sodium metabisulfite and stir until the solid is completely dissolved and the liquid is uniform without stratification.

[0076] Reaction system: Add 2L of purified water to the reactor, heat to 25±3℃, start stirring, add 23.4g of raw materials and 25.6g of sodium pyrosulfite and stir for 10min, let in air, adjust the pH to 7.3, add 2g of defoamer, confirm the temperature and pH, and add 800mL of the above-mentioned invertase solution to start the reaction. During the reaction, maintain the pH at 7.4 with a 2M NaOH aqueous solution until the reaction is completed. Add the substrate solution at the beginning of the reaction, and control the flow rate at 15mL / 10min.

[0077] The first enzyme supplementation time is about 9 hours, and 16g of sodium pyrosulfite and 400mL of the above-mentioned invertase solution are added. The second enzyme supplementation time is about 27 hours, which is determined according to the reaction situation. 16g of sodium pyrosulfite and 400mL of the above-mentioned invertase solution are added. After 42 hours of reaction, the substrate residue is 0% and the conversion rate reaches 100%.

[0078] Example 6

[0079] This example provides a method for biocatalytic synthesis of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate in a 350 L reaction system (500 L reactor).

[0080] Preparation of substrate solution: Add 22.87 kg of substrate (6,6-dimethyl-3-azabicyclo[3.1.0]hexane) and 144.38 kg of purified water into a container, stir for 15±5 minutes, then add 26.25 kg of sodium metabisulfite and stir until the solid is completely dissolved and the liquid is uniform without stratification.

[0081] Reaction system: Add 100 kg of purified water to the reactor, heat to 25 ± 3 ° C, start stirring, add 1.17 kg of raw materials, 1.28 kg of sodium pyrosulfite and stir for 10 minutes, let in air, adjust the pH to 7.3, add 100 g of defoamer, confirm the temperature and pH, and add 40 L of the above-mentioned invertase solution to start the reaction. During the reaction, the pH is maintained at 7.4 with a 2M NaOH aqueous solution until the reaction is completed. Add the substrate solution at the beginning of the reaction, and control the flow rate at 0.75 L / 10 min.

[0082] The first enzyme supplementation time is about 9 hours, and 0.8 kg sodium pyrosulfite and 20 L of the above-mentioned invertase solution are added. The second enzyme supplementation time is about 27 hours, which is determined according to the reaction situation, and 0.8 kg sodium pyrosulfite and 20 L of the above-mentioned invertase solution are added. After 42 hours of reaction, the substrate residue is 0% and the conversion rate reaches 100%.

[0083] Example 7

[0084] This example provides a method for biocatalytically synthesizing sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate in a 10.5T reaction system (15T reactor).

[0085] Preparation of substrate solution: Add 686.1 kg of substrate (6,6-dimethyl-3-azabicyclo[3.1.0]hexane) and 4331.4 kg of purified water into a container, stir for 15±5 minutes, then add 787.5 kg of sodium metabisulfite and stir until the solid is completely dissolved and the liquid is uniform without stratification.

[0086] Reaction system: Add 3T purified water to the reactor, heat to 25±3℃, start stirring, add 35.1kg raw material, 38.4kg sodium pyrosulfite and stir for 10min, introduce air, adjust pH to 7.3, add 3kg defoamer, confirm the temperature and pH, and add 1.2T of the above-mentioned invertase solution to start the reaction. During the reaction, maintain the pH at 7.4 with 2M NaOH aqueous solution until the reaction is completed. Add substrate solution at the beginning of the reaction, and control the flow rate at 22.5L / 10min.

[0087] The first enzyme supplementation time is about 9 hours, and 24 kg of sodium pyrosulfite and 600 L of the above-mentioned invertase solution are added. The second enzyme supplementation time is about 27 hours, which is determined according to the reaction situation. 24 kg of sodium pyrosulfite and 600 mL of the above-mentioned invertase solution are added. After 42 hours of reaction, the substrate residue is 0.02% and the conversion rate reaches 99.7%.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A monoamine oxidase, characterized in that: The amino acid sequences of the monoamine oxidase are shown in SEQ ID No. 15-19.

2. A polynucleotide encoding the monoamine oxidase according to claim 1.

3. A recombinant expression vector comprising the polynucleotide as shown in claim 2.

4. A monoamine oxidase engineered bacterium having the recombinant expression vector as claimed in claim 3.

5. A converting enzyme solution, which is obtained by fermenting, culturing, disrupting cells and flocculating the catalase engineered bacteria and the monoamine oxidase engineered bacteria according to claim 4, collecting the supernatants and mixing them in proportion.

6. The invertase solution according to claim 5, characterized in that: The catalase engineered bacteria comprises the amino acid sequence shown in SEQ ID No.

20.

7. A method for biocatalytic synthesis of sodium (1R, 2S, 5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate, characterized in that: In a liquid reaction system, 6,6-dimethyl-3-azabicyclo[3.1.0]hexane is used as a substrate and oxygen is used as an oxidant. The invertase solution of claim 5 is used to carry out a desymmetric reaction and simultaneously carry out an addition reaction with sodium pyrosulfite to generate the corresponding aminosulfonate.

8. The method according to claim 7, characterized in that: The substrate is added into the reaction system in a feeding manner, and the substrate concentration in the reaction system is controlled at 5-10 g / L.

9. The method according to claim 8, characterized in that: The amount of the invertase solution added is 20-25 v / v % of the total reaction system.

10. The method according to claim 9, characterized in that: The sodium pyrosulfite and the substrate are prepared into a solution according to a molar mass ratio of 1:1, and are added into the reaction system in a flow-feed manner together with the substrate.

11. The method according to claim 10, characterized in that: The enzyme catalytic reaction time is 36-48h, the reaction temperature is 25±3°C, and the substrate residue is monitored during the reaction.

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