A method for catalytically nitrogen-acetylating small-molecule nitrogen heterocyclic compounds

Abstract

A method for catalyzing the N-acetylation of small molecule nitrogen-containing heterocyclic compounds, belonging to the technical field of enzyme engineering research and development. Aiming at the problem that there is no existing technology for catalyzing the N-acetylation reaction of nitrogen-containing heterocycles, the present invention provides a method for efficiently catalyzing the acetylation reaction of nitrogen heterocycles by using a class of cutinases. Specifically, a plasmid modified with a cutinase gene is introduced into Escherichia coli BL-21 to obtain recombinant Escherichia coli, and then the recombinant Escherichia coli is cultured and induced. After steps such as cell disruption, a crude enzyme solution is obtained, and then through protein purification and concentration, a purified cutinase is obtained, which is added to an organic reagent containing an amino donor and an acyl donor for reaction. The method of the present invention uses cheap carboxylic acid compounds as raw materials and, for the first time, uses cutinase as a catalyst to prepare N-acetylated nitrogen heterocyclic derivatives, which can effectively bind hydrophilic nitrogen heterocycles and acetic acid, facilitating the recognition of nitrogen heterocyclic substrates with larger molecular structures by the active center, and is a green and safe preparation method.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering research and development, and particularly relates to a method for catalyzing the nitrogen acetylation of a small molecule nitrogen-containing heterocyclic compound. Background Art

[0002] Amide groups, as important functional groups in chemical building blocks, are present in many drug molecules, such as the tyrosine kinase inhibitor Imiatinib, the anti-HIV drug Dolutegravir, the cystic fibrosis drug Ivacaftor, the multiple sclerosis drug Terflunamide, and the antibiotic β-lactam. Over the past 40 years, the synthesis of amide bonds has accounted for 21.3% of all chemical reactions in drug synthesis. A 2011 survey of drugs developed by three major international pharmaceutical companies revealed that 54% of drug structures contained amide bonds, further demonstrating the importance of amide bonds. Currently, traditional chemical methods for synthesizing amides typically involve two or more steps. This process requires the activation of carboxylic acids with coupling reagents to form reactive intermediates such as acyl chlorides, anhydrides, and acyl azides. These reactive intermediates then undergo nucleophilic substitution with amines to form amides. However, this method suffers from low conversion rates and requires the use of numerous corrosive and toxic reagents, resulting in poor atom economy. For example, preparing 1 kg of the anti-AIDS drug Fuzeon (36 amino acids) requires 45 kg of raw materials, even excluding the organic reagents required for the synthesis and purification steps. In 2007, the American Chemical Society's Green Chemistry Institute voted to identify "efficient and economical synthesis of amides" as one of the top challenges in synthetic chemistry.

[0003] Currently, biocatalysis has been successfully applied to many key transformations, such as reduction, hydroxylation, oxidation, and hydrolysis, with some areas already industrialized. Cutinases, which belong to the α / β hydrolase class, possess the characteristic amino acid sequence of glycine (Gly)-tyrosine (Tyr)-serine (Ser)-glutamine (Gln)-glycine (Gly), along with the catalytic triad of serine (Ser)-histidine (His)-aspartate (Asp). Depending on their source, cutinases can be classified as either prokaryotic or eukaryotic. Eukaryotic cutinases generally have an optimum temperature between 30-40°C, while prokaryotic cutinases have a higher optimum temperature, generally between 50-60°C. Lipases, also belonging to the α / β hydrolase class, are characterized by a "lid" composed of one or more α-helices. This lid has hydrophilic and lipophilic properties, enabling lipases to activate the interface. Unlike lipases, most cutinases lack the "lid" found in their structure, leaving the key catalytic amino acid, serine, exposed to the solvent. Furthermore, the cutinase catalytic triad is typically located in a surface groove surrounded by hydrophobic amino acids, which facilitates substrate accommodation. This gives cutinases a wider substrate range, such as the polymerization and degradation of polyesters, and also opens up more possibilities for biocatalysis of nitrogen-containing heterocyclic nitrogen acetylation. Summary of the Invention

[0004] The present invention addresses the problem that there is no existing technology to catalyze the nitrogen acetylation reaction of nitrogen-containing heterocyclic rings, and provides an enzyme tool. The specific technical solution is as follows:

[0005] The present invention provides a method for catalyzing the nitrogen acetylation of a small molecule nitrogen-containing heterocyclic compound, the method comprising the following steps:

[0006] Step 1: Introduce the plasmid modified with cutinase gene into E. coli BL-21 to obtain recombinant E. coli, culture the recombinant E. coli, and when the bacterial solution OD 600 When the concentration is between 0.6 and 0.8, the fermentation liquid is induced, and the supernatant is discarded. The bacteria are resuspended in PBS buffer and the centrifugation step is repeated twice to obtain an E. coli BL21 suspension.

[0007] Step 2: Use a high-pressure cell disruptor to disrupt the cells, centrifuge, and discard the precipitate to obtain a crude enzyme solution; prepare the solvent required for protein purification, use a nickel column to purify the protein to obtain the target protein, and use an ultrafiltration tube to concentrate the protein to obtain the purified enzyme;

[0008] Step 3: Dissolve the amino donor and acyl donor in an organic solvent, add the purified enzyme to react, and perform gas phase quantitative detection.

[0009] In one embodiment of the present invention, the cutinase in step 1 is any one of the following cutinases:

[0010] Cutinase 1 having an amino acid sequence as shown in SEQ ID NO. 1;

[0011] Cutinase2 having an amino acid sequence as shown in SEQ ID NO. 2;

[0012] Cutinase3 having an amino acid sequence as shown in SEQ ID NO. 3;

[0013] Cutinase4 having an amino acid sequence as shown in SEQ ID NO. 4;

[0014] Cutinase5 having an amino acid sequence as shown in SEQ ID NO. 5;

[0015] The amino acid sequence of cutinase6 is shown in SEQ ID NO.6.

[0016] In one embodiment of the present invention, the culture in step 1 is cultured using LB-kana liquid culture medium at 37°C; the induction conditions are: adding IPTG to a final concentration of 0.5 mM and inducing at 16°C for 16 hours; and the centrifugation conditions are: centrifugation at 4°C and 8000 rpm for 5 minutes.

[0017] In one embodiment of the present invention, the centrifugation condition in step 2 is 4° C. and 13,500 rpm for 30 min.

[0018] In one embodiment of the present invention, the preparation method of the solvent required for protein purification in step 2 is as follows: accurately weigh 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 0.3404g imidazole in 500mL UP water to obtain Binding Buffer; accurately weigh 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 1.5318g imidazole in 500mL UP water to obtain Washing Buffer; accurately weigh 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 17.02g imidazole in 500mL UP water to obtain Elution Buffer.

[0019] In one embodiment of the present invention, the protein purification step in step 2 is: first, the column is equilibrated with 5 column volumes of Binding Buffer, the crude enzyme solution is repeatedly loaded three times, impurities are washed with 5 column volumes of Washing Buffer, and the target protein is collected with 3 column volumes of Elution Buffer.

[0020] In one embodiment of the present invention, the amino donor in step 3 includes but is not limited to piperidine, morpholine, tetrahydropyrrole, piperazine, and N-methylpiperazine.

[0021] In one embodiment of the present invention, the amino donor in step 3 further comprises a piperidine, morpholine, tetrahydropyrrole, piperazine, or N-methylpiperazine derivative modified with any one of hydroxyl, carbonyl, ester, halogen, alkyl, and aryl substituents.

[0022] In one embodiment of the present invention, the acyl donor in step 3 is a carboxyl-modified compound, including but not limited to acetic acid, propionic acid, and butyric acid; the molar ratio of the amino donor to the acyl donor is 1:1.1-5.

[0023] In one embodiment of the present invention, the organic solvent in step 3 is any one of acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, n-hexane, and water, or a mixture of two or more thereof.

[0024] In one embodiment of the present invention, the final concentration of the purified enzyme in step 3 is 0.1-5 mg / mL; the reaction temperature is 15-60° C., and the reaction time is 0.5-72 h.

[0025] In one embodiment of the present invention, the gas phase detection conditions in step 3 are as follows: the gas chromatograph uses an Agilent 8890GC system, the gas phase detection conditions are: 30m×250μm×250μm HP-INNOWAX chromatographic column, heater 220°C, air flow rate 400mL / min, hydrogen fuel gas flow rate 30mL / min, tail gas (N2) flow rate 25mL / min. The column oven temperature is a maximum of 270°C, and the column oven temperature gradient is set as follows: initial temperature 80°C, 15°C / min to 200°C, then hold for 5 minutes, 20°C / min to 240°C, and hold for 5 minutes.

[0026] Beneficial effects of the present invention:

[0027] The present invention addresses the problem that there is no enzyme in the prior art to catalyze the nitrogen acetylation reaction of nitrogen-containing heterocycles. The present invention provides a method for efficiently catalyzing the acetylation reaction of nitrogen-containing heterocycles using a type of cutinase, which has the following advantages:

[0028] (1) The present invention selects cheap carboxylic acid compounds as raw materials and uses cutinase as a catalyst for the first time to prepare acetyl-modified nitrogen heterocyclic derivatives, which is a green and safe preparation method.

[0029] (2) The cutinase used in the present invention is a serine-histidine-aspartate catalytic triad with the characteristics of α-β serine hydrolase, which can efficiently catalyze the dehydration reaction; at the same time, it does not have the "lid structure" characteristic of lipase-like α-β serine hydrolase, which can effectively bind to hydrophilic nitrogen heterocycles and acetic acid, which is conducive to the recognition of nitrogen heterocycle substrates with larger molecular structures and the active center. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of the gas phase detection results of the reaction liquid obtained in Example 1. DETAILED DESCRIPTION

[0031] The present invention aims to provide a method for catalyzing the nitrogen acetylation of small molecule nitrogen-containing heterocyclic compounds. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0032] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0033] The LB-kana liquid culture medium used in the present invention is prepared as follows: 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, 1 mL of 1 mol / L sodium hydroxide aqueous solution adjusted to pH 7.4, kanamycin is added to a final concentration of 50 μg / mL, the volume is adjusted to 1 L with deionized water, and steam sterilized under high pressure for 20 min.

[0034] The gas phase detection conditions involved in the present invention are: gas chromatography using an Agilent 8890GC system, gas phase detection conditions: 30m×250μm×250μm HP-INNOWAX chromatographic column, heater at 220°C, air flow rate of 400mL / min, hydrogen fuel flow rate of 30mL / min, tail gas (N2) flow rate of 25mL / min. The column oven temperature is set to a maximum of 270°C, and the column oven temperature gradient is set as follows: initial temperature 80°C, heating at 15°C / min to 200°C, holding for 5 minutes, heating at 20°C / min to 240°C, and holding for 5 minutes.

[0035] The preparation method of the solvent required for protein purification of the present invention is as follows: accurately weighing 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 0.3404g imidazole in 500mL UP water to obtain Binding Buffer; accurately weighing 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 1.5318g imidazole in 500mL UP water to obtain Washing Buffer; accurately weighing 8.95g Na2HPO4·12H2O, 8.766g NaCl, and 17.02g imidazole in 500mL UP water to obtain Elution Buffer.

[0036] Yield calculation formula:

[0037] Yield = [C(acetylated product) × V / M(acetylated product)] / [C(amino donor) × V / M(amino donor)]

[0038] C: Liquid phase determination of the concentration of the raw material amino donor and the product acetylated product, g / L; V: Reaction liquid volume, L; M: Molar molecular weight of the raw material amino donor and the product acetylated product.

[0039] Example 1:

[0040] The amino acid sequence of cutinase-1 used in this example is shown in SEQ ID NO. 1: MNLRLLTLALAAVAAASPVDIQERQLSGGNELRDGSCKPITFIFARASTEPGLLGISTGPAVCNGLKMAKAGQVACQGVGPKYTADLASNALPENTSPAAIQEAQDLFQQAVTKCPDTQIVAGGYSQGTAVMDDSIKRLPDNVKEKIKGVVLFGYTRNAQEHGQIANFPKDKVKVYCAVGDMVCDGTLIVGPAHFTYLGNTGEATQFLLGYLRFLQLTQLP

[0041] The cutinase is disclosed in Genomesequencing and analysis of Aspergillusoryzae, By Machida, Masayuki; Asai, Kiyoshi; Sano, Motoaki; Tanaka, Toshihiro; Kumagai, Toshitaka; Te Rai, Goro; Kusumoto, Ken-Ichi; Arima, Toshihide; Akita, Osamu; Kashiwagi, Yutaka; etal, FromNature (London, United Kingdom) (2005), 438 (7071), 1157-1161.

[0042] Step 1: The cutinase-1 modified plasmid pET-28a-cutinase-1 was introduced into Escherichia coli BL21, and the obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0043] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-1 is obtained, and the protein concentration is measured using the Bradford method.

[0044] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (0.75 g / L piperazine and 1:3 molar ratio of piperazine to acetic acid). Add 30 μL of cutinase-1 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. Filter the reaction mixture through a membrane and analyze using an Agilent 8890 GC. Figure 1 In the figure, 7.117 min is the gas phase peak result of 1-acetylpiperazine, and the yield of 1-acetylpiperazine is measured to be 21.84%.

[0045] Example 2:

[0046] The amino acid sequence of cutinase-2 used in this example is shown in SEQ ID NO. 2: MNYKLLTLALASLAAANPIERQRKIHPAVLSGGDELRNGDCQPVTFIFARASTEQGLLGGSTGPALCNRLKSALDGVACQGVGPKYQATLAANALPKGTSDEAIEEAQSLFQLAAEKCPDTQIVAGGYSQGTAVMHGAIPGLDDALKDKIKGVVLFGDTRNQQDNGQIPDFPKDKIKIYCAAGDMVCYGTLIVAAPHFSYIADVPDAADFLVSKLE

[0047] The enzyme is disclosed in sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae, By Galagan, James E.; Calvo, Sarah E.; Cuomo, Christina; Ma, Li-Jun; Wortman, Jen nifer R.; Batzoglou, Serafim; Lee, Su-In; Bastuerkmen, Meray; Spevak, Christina C.; Clutterbuck, John; et al., From Nature (London, United Kingdom) (2005), 438 (7071), 1105-1115.

[0048] Step 1: The cutinase-2 modified plasmid pET-28a-cutinase-2 was introduced into Escherichia coli BL21. The obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0049] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-2 is obtained, and the protein concentration is measured using the Bradford method.

[0050] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. Add 30 μL of cutinase-2 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 24.86%.

[0051] Example 3:

[0052] The amino acid sequence of cutinase-3 used in this example is shown in SEQ ID NO. 3: MNLRLLTLALAGLAAASPVAIQERQFSSGNELRDGACKPITFIFARASTEPGLLGMSTGPAVCNNLKAAKPGQVACQGVGPAYTADLGSNALPENTSPAAINEAVDLFKQAASKCPDTQIVAGGYSQGTAVMDGSIKRLPDEVKEKIKGVVLFGYTRNAQERGQIANFPKDKVKIYCAMGDLVCDGTLIVTAAHFTYGANTGDAARFLLGKLSTA

[0053] The enzyme is disclosed in Genomic Islands in the pathogenic filamentous fungus Aspergillus fumigatus, By Fedorova Natalie D; Khaldi Nora; Joardar Vinita S; Maiti Rama; Amedeo Paolo; Anderson Michael J; Crabtree Jonathan; SilvaJoana C; Badger Jonathan H; Albarraq Ahmed; PLoSgenetics(2008),4(4),e1000046.

[0054] Step 1: The cutinase-3 modified plasmid pET-28a-cutinase-3 was introduced into Escherichia coli BL21. The obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0055] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-3 is obtained, and the protein concentration is measured using the Bradford method.

[0056] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. Add 30 μL of cutinase-3 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 27.19%.

[0057] Example 4:

[0058] The amino acid sequence of cutinase-4 used in this example is shown in SEQ ID NO.4: MSLRSLFVAGLATLALAVPAPQIQARQGMSSNELESGPCRDVTFIFARGSTEQGNMGLIVGPGVCSSLKKDLGSDKVACQGVGGAYTAQLAPNFLSQNTNQASINAATDMFDLANTKCPNTKIVAGGYSQGSAVIDNTIQALGSDLKAKVKGVVLFGFTRNVADKGQIPGYPKDQTKIYCAVGDMVCVNTLIITPAHLTYGADAGDAAKFLASKVQE

[0059] The enzyme is disclosed in Genomic Islands in the pathogenic filamentous fungus Aspergillus fumigatus, By Fedorova Natalie D; Khaldi Nora; Joardar Vinita S; Maiti Rama; Amedeo Paolo; Anderson Michael J; Crabtree Jonathan; SilvaJoana C; Badger Jonathan H; Albarraq Ahmed; PLoSgenetics(2008),4(4),e1000046.

[0060] Step 1: The cutinase-4 modified plasmid pET-28a-cutinase-4 was introduced into Escherichia coli BL21. The obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0061] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-4 is obtained, and the protein concentration is measured using the Bradford method.

[0062] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. Add 30 μL of cutinase-4 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 26.50%.

[0063] Example 5:

[0064] The amino acid sequence of cutinase-5 used in this example is shown in SEQ ID NO.5: MNLRLLTLALAGLAAASPVAIEERQLSSGNELRNGACKPITFIFARASTEPGLMGISTGPAVCNSLKAAKPGQVACQGVGPAYTADLASNALPENTSQAAINEAMELFKQAASKCPDTQIVAGGYSQGTAVMDGSIKRLPEEVKERIKGVVLFGYTRNAQERGQIANFPKDKVKIYCAMGDLVCDGTLIVTAAHFTYGANTGDAARFLLGKLTA

[0065] The enzyme is disclosed in PrevalenceandmechanismsofazoleresistanceinclinicalisolatesofAspergillussectionFumigatispeciesinaCanadiantertiarycarecentre,2000to2013,By Parent-Michaud, Maxime; Dufresne, Philippe J.; Fournier, Eric; Folch, Benjamin; Martineau, Christine; Moreira, Sandrine; Doucet, Nicolas; DeRepentigny, Louis; Dufresne, Simon F. From Journal of Antimicrobial Chemotherapy (2020), 75(4), 849-858.

[0066] Step 1: The cutinase-5 modified plasmid pET-28a-cutinase-5 was introduced into Escherichia coli BL21. The obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0067] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-5 is obtained, and the protein concentration is measured using the Bradford method.

[0068] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. Add 30 μL of cutinase-5 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 20.31%.

[0069] Example 6:

[0070] The amino acid sequence of cutinase-6 used in this example is shown in SEQ ID NO.6: MKFSIISTLFAATASALPAGQDAAALEARQLGGSITRNDLANGNSGSCPGVIFIYARGSTEAGNLGTLGPRVASKLEAKYGKNGVWIQGVGGAYRATLGDNALPRGTSSAAIREMLGHFNDANQKCPDAVLIAGGYSQGAALAAASVTDVDASIREKIAGVVLFGYTKNLQNRGKIPSYPEDRTKVFCNTGDLVCTGSLIVAAPHLAYQSDASNGAPEFLIQKADAAGAA

[0071] The enzyme is disclosed in Deciphering the cryptogenome:genome-wide analyse of the rice pathogen Fusarium fujikuroireveal complex regulation of secondary metabolism and novel metabolites,

[0072] By Wiemann, Philipp; Sieber, Christian M. K.; von Bargen, Katharina W.; Studt, Lena; Niehaus, Eva-Maria; Espino, Jose J.; Hus s,Kathleen;Michielse,CarolineB.;Albermann,Sabine;Wagner,Dominik;etal,FromPLoSPathogens(2013),9(6),e1003475.

[0073] Step 1: The cutinase-6 modified plasmid pET-28a-cutinase-6 was introduced into Escherichia coli BL21. The obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0074] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-6 is obtained, and the protein concentration is measured using the Bradford method.

[0075] Step 3: Dissolve piperazine and acetic acid in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. Add 30 μL of cutinase-4 to a final enzyme concentration of 0.9 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 22.89%.

[0076] Comparative Example 1:

[0077] Piperazine and acetic acid were dissolved in 1 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperazine concentration of 0.75 g / L and a molar ratio of 1:3. 30 μL of PBS buffer was added and the reaction was catalyzed at 40°C for 12 hours. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperazine was 17.51%.

[0078] From the data of Examples 1-6 and Comparative Example 1, it can be seen that the six cutinases, cutinase-1 to cutinase-6, have a transacetylating biocatalytic effect on nitrogen-containing heterocycles.

[0079] Example 7:

[0080] Step 1: The cutinase-1 modified plasmid pET-28a-cutinase-1 was introduced into Escherichia coli BL21, and the obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0081] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-1 is obtained, and the protein concentration is measured using the Bradford method.

[0082] Step 3: Dissolve piperidine and acetic acid in 5 mL of N,N-dimethylformamide (N,N-dimethylformamide) at a piperidine concentration of 3 g / L and a piperidine:acetic acid molar ratio of 1.5:2. Add cutinase-1 to a final enzyme concentration of 1.5 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperidine was 49.27%.

[0083] Comparative Example 2:

[0084] Piperidine and acetic acid were dissolved in 5 mL of N,N-dimethylformamide (DNFA) at a piperidine concentration of 3 g / L and a molar ratio of 1.5:2. Cutinase was added to a PBS buffer solution to a final PBS concentration of 1.5 mg / mL. The reaction was incubated at 40°C for 12 hours. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetylpiperidine was determined to be 35.23% based on the product standard curve.

[0085] From the data of Example 7 and Comparative Example 2, it can be seen that the six cutinases cutinase-1 to cutinase-6 have a transacetylating biocatalytic effect on nitrogen-containing heterocycles.

[0086] Example 8:

[0087] Step 1: The cutinase-1 modified plasmid pET-28a-cutinase-1 was introduced into Escherichia coli BL21, and the obtained positive transformants were inoculated into LB-kana liquid medium and cultured at 37°C until OD 600 Add IPTG to a final concentration of 0.5 mM at a concentration between 0.6 and 0.8, and induce at 16°C for 16 hours; centrifuge and discard the supernatant. The centrifugation conditions are: 4°C, 8000 rpm for 5 minutes; wash the bacteria with PBS and resuspend.

[0088] Step 2: Disrupt the cells with a high-pressure cell disruptor and centrifuge again to discard the precipitate to obtain a crude enzyme solution. The centrifugation conditions are 4°C and 13,500 rpm for 30 min. Use 5 column volumes of Binding Buffer to equilibrate the nickel column, repeatedly load the crude enzyme solution three times, use 5 column volumes of Washing Buffer to wash away impurities, and use 3 column volumes of Elution Buffer to collect the target protein. Finally, the purified enzyme cutinase-1 is obtained, and the protein concentration is measured using the Bradford method.

[0089] Step 3: Dissolve tetrahydropyrrole and acetic acid in 5 mL of acetonitrile to a tetrahydropyrrole concentration of 3 g / L and a tetrahydropyrrole:acetic acid molar ratio of 1.5:2. Add cutinase-1 to a final enzyme concentration of 1.5 mg / mL. Incubate at 40°C for 12 h. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetyltetrahydropyrrole was 38.01%.

[0090] Comparative Example 3:

[0091] Tetrahydropyrrole and acetic acid were dissolved in 5 mL of acetonitrile to a 3 g / L tetrahydropyrrole concentration and a 1.5:2 molar ratio. Cutinase was added to a PBS buffer solution to a final PBS concentration of 1.5 mg / mL. The reaction was incubated at 40°C for 12 hours. The reaction mixture was filtered through a membrane and analyzed using an Agilent 8890 GC. The yield of 1-acetyltetrahydropyrrole was determined to be 29.45% based on the product standard curve.

[0092] From the data of Example 8 and Comparative Example 3, it can be seen that the six cutinases cutinase-1 to cutinase-6 have a transacetylating biocatalytic effect on nitrogen-containing heterocycles.

[0093] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for catalytic N-acetylation of small molecule nitrogen-containing heterocyclic compounds, characterized in that, the method comprises the following steps: S1. Introduce the plasmid modified with the cutinase gene into Escherichia coli BL-21 to obtain recombinant Escherichia coli. Culture the recombinant Escherichia coli. When the OD of the bacterial liquid is 600 between 0.6 and 0.8, perform induction. After induction, centrifuge the fermentation broth, discard the supernatant, resuspend the cells with PBS buffer, and repeat the centrifugation step twice to obtain an Escherichia coli BL21 suspension; the cutinase is cutinase1 with the amino acid sequence shown in SEQ ID NO.1; S2. Use a high-pressure cell disruptor to disrupt the bacterial cells, centrifuge, discard the precipitate, and obtain a crude enzyme solution; prepare the solvents required for protein purification, use a nickel column for protein purification to obtain the target protein, and use an ultrafiltration tube for concentration to obtain the purified enzyme; S3. Dissolve the amino donor and acyl donor in an organic solvent, add the purified enzyme for reaction, and perform gas-phase quantitative detection; the amino donor is piperazine, piperidine or pyrrolidine; when the amino donor is piperidine, dissolve piperidine and acetic acid in N,N-dimethylformamide, when the amino donor is piperazine, dissolve piperazine and acetic acid in N,N-dimethylformamide, and when the amino donor is pyrrolidine, dissolve pyrrolidine and acetic acid in acetonitrile; the molar ratio of the amino donor to the acyl donor is 1.5:2 to 1:3, and the final concentration of the purified enzyme is 0.9 to 1.5 mg / mL; the reaction temperature is 40 °C and the reaction time is 12 h.

2. The method according to claim 1, characterized in that, the cultivation in S1 is carried out using LB-kana liquid medium at 37 °C; the induction condition is: add IPTG with a final concentration of 0.5 mM and induce at 16 °C for 16 h; the centrifugation condition is: 4 °C, 8000 rpm, centrifuge for 5 min.

3. The method according to claim 1, characterized in that, the centrifugation condition in S2 is 4 °C, 13500 rpm, centrifuge for 30 min.

4. The method according to claim 1, characterized in that, The preparation method of the solvent required for purifying the protein described in S2 is as follows: Accurately weigh 8.95 g of Na 2 HPO 4 ·12H 2 O, 8.766 g of NaCl, and 0.3404 g of imidazole in 500 mL of UP water to obtain Binding Buffer; Accurately weigh 8.95 g of Na 2 HPO 4 ·12H 2 O, 8.766 g of NaCl, and 1.5318 g of imidazole in 500 mL of UP water to obtain Washing Buffer; Accurately weigh 8.95 g of Na 2 HPO 4 ·12H 2 O, 8.766 g of NaCl, and 17.02 g of imidazole in 500 mL of UP water to obtain Elution Buffer.

5. The method according to claim 4, characterized in that, the protein purification step in S2 is: first balance the column with 5 column volumes of Binding Buffer, and load the crude enzyme solution repeatedly three times; wash the miscellaneous proteins with 5 column volumes of Washing Buffer; collect the target protein with 3 column volumes of Elution Buffer.

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