A bifunctional enzyme PAI2 with CN-N bond and N-N bond synthesis capabilities, its preparation method and applications

Abstract

This invention relates to a bifunctional enzyme, PAI2, capable of synthesizing both C-N and N-N bonds, along with its preparation method and applications, belonging to the fields of biochemistry and synthetic biology. To address the problems in existing technologies for synthesizing proline and azaproline, a novel bifunctional enzyme, PAI2, is provided, capable of synthesizing both C-N and N-N bonds simultaneously. Furthermore, an engineered bacterium overexpressing the bifunctional enzyme PAI2 was successfully constructed using genetic engineering. Through induction of the engineered bacterium, high-purity, active bifunctional enzyme PAI2 was obtained, ultimately achieving the technical effect of simultaneously synthesizing proline and azaproline under the catalysis of the bifunctional enzyme PAI2.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry and synthetic biology, specifically relating to a bifunctional enzyme PAI2 with CN and NN bond synthesis capabilities, its preparation method, and its applications. Background Technology

[0002] Nitrogen-containing heterocyclic compounds are a very important class of organic synthesis intermediates, including pyridine containing a single nitrogen atom and pyrazole, piperazine, triazole, etc. containing multiple nitrogen atoms. They are the basic skeletons of many natural products with important physiological activities such as anti-inflammatory, antitumor, and antibacterial activity (WANG S, ZHANG B, CHEN J, et al. Recent Progress in Synthesis of Polysubstituted Pyrazoles[J]. Chinese Journal of Organic Chemistry, 2020, 40(1):). Among them, proline is one of the important amino acids for synthesizing human proteins and is also an important intermediate for synthesizing first-line antihypertensive drugs such as captopril and enalapril. Proline derivative azaproline is an important substrate in biologically important peptide sequences related to metallopeptidase activity and in organic catalysis (DUTTAGUPTA I, GOSWAMI K, SINHA S. Synthesis of cyclic α-hydrazino acids[J]. Tetrahedron, 2012, 68(39): 8347-57.). However, although the synthesis of proline has been industrialized, the synthesis of azaproline is very difficult. Only a few synthesized δ-azaproline (VOSS E, ARRAULT A, BODIGUEL J, et al. Efficient synthesis of enantiomerically pure(S)-delta-azaproline starting from(R)-alpha-hydroxy-gamma-butyrolactone via the Mitsunobu reaction[J]. Tetrahedron-Asymmetry, 2009, 20(15): 1809-12.) or substituted azaproline (GUERRA FM, MISH MR, CARREIRA E M. Versatile, diastereoselective additions of silyl ketene acetals, allyl tributylstannane, and Me3SiCN to N-acyl pyrazolines: Asymmetric synthesis of densely functionalized pyrazolidines[J]. Organic Letters, 2000, 2(26): 4265-7.)The synthesis of nitrogen-containing heterocyclic compounds mainly involves the formation of CN or NN bonds. Therefore, the importance of developing effective synthetic strategies to generate CN and NN bonds is beyond doubt.

[0003] In recent decades, the development of transition metal catalysts has made them important tools for the synthesis of CN bonds. However, traditional methods inevitably suffer from problems such as expensive catalysts and metal residues that urgently need to be addressed. Furthermore, the complex reaction steps and limited substrate diversity have hindered the widespread application of this method. In the chemical synthesis of compounds containing N-N bonds, although existing research has shown that intramolecular or intermolecular N-N bonds can be constructed through various methods (GUO Q, LU Z. Recent Advances in Nitrogen–Nitrogen Bond Formation[J]. Synthesis, 2017, 49(17):3835-47.), the synthesis of N-N bonds often uses hydrazine hydrate as a raw material, such as 1,2,4-triazol-5-one and benzoyl hydrazine (NAMMALWARB, MUDDALA NP, WATTS FM, et al. Efficient conversion of acids and esters toamides and transamidation of primary amides using OSU-6[J]. Tetrahedron, 2015, 71(48):9101-11.). These synthetic methods are simple and effective, but the raw material hydrazine hydrate used in them is a highly toxic substance, corrosive, and prone to explosion, posing a problem of raw material sustainability. The synthesis of other N-N bond compounds, such as melamine, requires the substrate urea to react under high temperature and pressure conditions of 0.5-7 MPa and 380-400℃, which poses certain risks. Therefore, it is still necessary to continuously search for or optimize existing chemical synthesis routes, such as free radical reactions of C(sp2)-H bonds in transition metal-free systems (WU Y, CHEN J, LI Q, et al. Progress in C—N Bond Formation Involving C(sp2)—HBond through Transition-Metal-Free Radical Reactions[J]. Chinese Journal of Organic Chemistry, 2020, 40(3):), or to develop green and environmentally friendly biosynthetic routes that can be conveniently carried out under mild conditions using readily available raw materials and inexpensive catalysts.

[0004] However, in the biosynthetic pathways of nitrogen-containing natural products, only a few examples of direct CH bond amination catalyzed by cytochrome P450 to form Indolactam V have been elucidated, such as LtxB (HUYNH MU, ELSTON MC, HERNANDEZ NM, et al. Enzymatic Production of (-)-Indolactam V by LtxB, a Cytochrome P450 Monooxygenase[J]. J Nat Prod, 2010, 73(1):71-4.), TleB (HE F, MORIT, MORITA I, et al. Molecular basis for the P450-catalyzed CN bond formation in indolactam biosynthesis[J]. Nat Chem Biol, 2019, 15(12):1206-13.), and HinD (WEI J, LIUY. Mechanistic Insights into the P450 TleB-Catalyzed Unusual Intramolecular CN). Bond Formation Involved in the Biosynthesis of Indolactam V[J]. J Chem Inf Model, 2021, 61(7): 3638-48.), etc., the catalytic mechanism and protein structure have been elucidated. However, more than ten N-N bond synthases have been reported, including KtzT (DU YL, HE HY, HIGGINS MA, et al. A heme-dependent enzyme forms the nitrogen-nitrogen bond in piperazate[J]. Nat Chem Biol, 2017, 13(8): 836-+.), MatF (LEIPOLDT F, SANTOS-ABERTURAS J, STEGMANND P, et al. Warhead biosynthesis and the origin of structural diversity in hydroxamate metalloproteinase inhibitors[J]. Nature Communications, 2017, 8(1):), CreM (WALDMAN AJ, BALSKUS E P.), etc.在克瑞霉素生物合成中发现重氮形成酶[J].《有机化学杂志》,2018年,第83卷(第14期):7539 - 46页)、肼合酶(卡尔塔尔B,马尔克W J,德阿尔梅达N M等.厌氧氨氧化的分子机制[J].《自然》,2011年,第479卷(第7371期):127 - 30页)、细胞色素P460(CYP460)(卡兰托J D,维尔贝A C,兰卡斯特K M.欧洲亚硝化单胞菌细胞色素P460是硝化作用与一氧化二氮排放之间的直接联系[J].《美国国家科学院院刊》,2016年,第113卷(第51期):14704 - 14709页)和CYP450nor(麦夸特斯A B,维尔高N E,莱纳特N.真菌细胞色素P450一氧化氮还原酶(P450nor)关键中间体的模型配合物[J].《化学生物学当前观点》,2014年,第19卷(第82 - 9页),重排反应包类型的Spb40(松田K,富田T,筱屋K等.细菌中前所未有的肼形成机制的发现[J].《美国化学会志》,2018年,第140卷(第29期):9083 - 9086页)、PyrN(赵G,姚S,罗斯柴尔德K W等.吡唑霉素的生物合成基因簇——一种具有罕见吡唑部分的C -核苷抗生素[J].《化学生物化学》,2019年,第21卷(第5期):644 - 649页)、SznF(何H - Y,亨德森A C,杜Y - L等.Two-Enzyme Pathway Linksl-Arginine to Nitric Oxide in N-Nitroso Biosynthesis[J]. J Am Chem Soc, 2019, 141(9): 4026-33.)(NG TL, ROHAC R, MITCHELL AJ, et al. An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin[J]. Nature, 2019, 566(7742): 94-9.), GrbD(HERMENAU R, MEHL JL, ISHIDA K, et al. Genomics-Driven Discovery of NO-Donating Diazeniumdiolate Siderophores in Diverse Plant-Associated Bacteria[J]. Angewandte Chemie International Edition, 2019, 58(37): 13024-9.), etc., but their catalytic mechanism has not yet been elucidated, limiting research on the biosynthesis of nitrogen-containing compounds. Currently, the number of discovered CN-bond synthases and NN-bond synthases is small, and no bifunctional enzymes capable of simultaneously catalyzing CN-bonds and NN-bonds have been reported, highlighting the urgent need for their development. Summary of the Invention

[0005] To address the problems existing in the synthesis of proline and azaproline using current technologies, this invention provides a bifunctional enzyme PAI2 with the ability to synthesize CN and NN bonds. The amino acid sequence of the bifunctional enzyme PAI2 is shown in SEQ ID NO.1.

[0006] The present invention also provides a gene encoding the above-mentioned bifunctional enzyme PAI2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides plasmids carrying the above-mentioned genes.

[0008] The present invention also provides recombinant cells expressing the above-mentioned bifunctional enzyme PAI2 or carrying the above-mentioned gene.

[0009] This invention also provides a method for constructing engineered bacteria that overexpress the bifunctional enzyme PAI2. The target gene pai2 is cloned into the vector pET22b using Nde I and Xho I restriction sites, and a His tag is added to obtain the recombinant plasmid pET22b-HTPAI2. pET22b-HTPAI2, along with the molecular chaperone plasmid pG-KJE8, is then transformed into competent cells of *Escherichia coli* BL21(DE3) using a heat shock method. A single colony of the recombinant strain is then obtained, which is the engineered bacteria.

[0010] This invention also provides a method for preparing the above-mentioned bifunctional enzyme PAI2, specifically comprising the following steps:

[0011] (1) Obtaining crude enzyme solution: Add 125 μL of 1M IPTG, 2.5 mL of inducer 1, and 5 μL of inducer 2 to the above-mentioned engineered bacterial culture. Induce at 16℃ for 20 h, centrifuge, collect cells, resuspend in lysis buffer, autoclave, centrifuge, and collect the supernatant to obtain the crude enzyme solution of PAI2. The composition of inducer 1 is 0.1M 5-aminolevulinate, 0.1M ferrous ammonium sulfate, 50 mg / mL arabinose, and the remainder is water. The composition of inducer 2 is 0.25 mg / mL tetracycline, and the remainder is water.

[0012] (2) Purification of crude enzyme solution: His-labeled protein was separated by Ni-NTA resin. After washing the protein with washing buffer, the protein was eluted with elution buffer. Then, the protein was dialyzed three times with storage buffer to remove imidazole in the buffer. Heme chloride was added to make the ratio of heme to enzyme 2:1. After incubation for 12 hours, the protein was dialyzed three times to remove excess heme.

[0013] Further specifying, the method for preparing the engineered bacterial culture in step (1) involves inoculating the engineered bacteria into LB medium containing antibiotics to obtain a seed culture, and then inoculating the seed culture into LB medium containing antibiotics at an inoculation rate of 1%, and culturing until OD. 600 It is 0.6.

[0014] Further specifying, the washing buffer in step (2) consists of 300mM NaCl, 50mM Tris-HCl, 50mM imidazole, with the remainder being water, and pH 8.0; the elution buffer consists of 300mM NaCl, 50mM Tris-HCl, 250mM imidazole, with the remainder being water, and pH 8.0; the storage buffer consists of 300mM NaCl, 50mM Tris-HCl, with the remainder being water, and pH 8.0.

[0015] This invention also provides applications of the aforementioned bifunctional enzyme PAI2, in the context of LN 4Using -OH-diaminobutyric acid and 2-amino-5-hydroxyvalerate as substrates, the bifunctional enzyme PAI2 is added to catalyze the production of azaproline and proline.

[0016] Further specified, the concentration of the substrate is 1-10 mM, the amount of the bifunctional enzyme PAI2 added is 0.087 U / g substrate to 0.879 U / g substrate; the catalysis is carried out at 26℃-37℃ for 1-6 h.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention provides a novel and unique bifunctional enzyme PAI2 that simultaneously possesses the ability to synthesize CN bonds and NN bonds, thereby obtaining an active biocatalytic mechanism, expanding the family of CN bond synthases and NN bond synthases, and obtaining engineered bacteria that overexpress the bifunctional enzyme PAI2 through the construction of recombinant vectors.

[0019] 2. This invention obtains high-purity bifunctional enzyme PAI2 by inducing and purifying engineered bacteria that overexpress the bifunctional enzyme PAI2.

[0020] 3. This invention provides a method for in vitro catalysis of LN using the bifunctional enzyme PAI2. 4 The method for synthesizing proline and azaproline from -OH-diaminobutyric acid and 2-amino-5-hydroxyvalerate solves the problems existing in the synthesis of CN and NN bonds in the prior art. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the pET22b-HTPAI2 recombinant plasmid structure;

[0022] Figure 2 The image shows the SDS-PAGE analysis results of PAI2 co-expressed with the molecular chaperone. Figure 2 a) in the figure shows the purification results of PAI2 without a molecular chaperone; Figure 2 Figure b) shows the purification results of PAI2 co-expressed with the molecular chaperone; Figure 2 In a) and b), M, P, S, F, W and E represent protein markers, precipitate, supernatant, flow-through, washing and elution fractions, respectively;

[0023] Figure 3 The image shows the LC-MS results of the in vitro catalytic reaction of 2-amino-5-hydroxyvalerate, where... Figure 3 a) in this context refers to the in vitro reaction and derivatization pathway of 2-amino-5-hydroxyvalerate. Figure 3 b) in the figure shows the LC-MS analysis results, with mass spectrometry information corresponding to the products;

[0024] Figure 4 LN 4 The LC-MS results of the in vitro catalytic reaction of -OH-diaminobutyric acid are shown in the figure. Figure 4 a) in the example is LN 4 In vitro reaction and derivatization pathway of -OH-diaminobutyric acid; Figure 4 b) in the figure shows the LC-MS analysis results, where the mass spectrometry information corresponds to the predicted molecular weight of the product.

[0025] Definitions and abbreviations:

[0026] Isopropyl galactothioglycoside: IPTG

[0027] Escherichia coli: E. coli

[0028] 5-Aminolevulinic acid: 5-ALA

[0029] 9-fluorenylmethoxycarbonyl chloride: Fmoc-Cl

[0030] Liquid chromatography-mass spectrometry (LC-MS)

[0031] LN 4 -OH-diaminobutyric acid: LN 4 -OH-Dab

[0032] Proline: Pro

[0033] Azaproline: Aza-Pro Detailed Implementation

[0034] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional techniques. Unless otherwise specified, all materials and reagents used in the following examples are commercially available; E. coli competent cells were purchased from TransGen Biotech Ltd.; all culture media were prepared with deionized water unless otherwise specified.

[0036] The reagents required for the preferred specific implementation are as follows:

[0037] LB medium: 5 g / L yeast extract, 10 g / L NaCl, 10 g / L peptone, sterilized at 121°C for 20 min.

[0038] 50 mg / mL kanamycin sulfate solution: Weigh 0.5 g of kanamycin sulfate solid and dissolve it in 10 mL of water.

[0039] 25 mg / mL chloramphenicol solution: Weigh 0.5 g of chloramphenicol solid and dissolve it in 20 mL of anhydrous ethanol.

[0040] 1M IPTG: 238g / mol × 1mol / L × 0.01L = 2.38g, that is, weigh 2.38g of IPTG powder and dissolve it in 10mL of water.

[0041] Inducer 1 (0.1M 5-aminolevulinate + 0.1M ferrous ammonium sulfate + 50mg / mL arabinose): Weigh 0.168g 5-aminolevulinate, 0.284g ferrous ammonium sulfate, and 0.5g arabinose, and dissolve them in 10mL of water.

[0042] Inducer 2 (0.25 mg / mL tetracycline): Weigh 0.0025 g of tetracycline and dissolve it in 10 ml of water.

[0043] Example 1: Construction of engineered bacteria overexpressing the bifunctional enzyme PAI2

[0044] This invention obtained a bifunctional enzyme, PAI2 (GenBank: QCX80659.1, derived from Streptomyces sp. YIM 121038), through gene mining, which possesses both N-N bond and CN bond synthesis functions. The amino acid sequence of this enzyme is shown in SEQ ID NO.1, and it has the highest amino acid sequence similarity (64.81%) to the previously reported N-N bond synthase KtzT (GenBank: ABV56600.1, derived from Kutzneria sp. 744). The nucleotide sequence encoding this bifunctional enzyme PAI2 is shown in SEQ ID NO.2.

[0045] SEQ ID NO.1:

[0046] MFVPRVYREPEESWKIDLVRGNPLGQLVSNGAEGEAPWVTHVPIIIDPRVTEPVTSLSGTTLWGHMNIGNPHWRALGPATPVAVTFSGPHAYVSPTVYETRPAAPTWNFTAVHIAGV LRKVDSTDETLATVQETVRAYEREFGADWSMTESIEYFRRILPGVGAFRIAISLADGMFKLSQEQPPHVRERVRASFACEASTAHREVAALMGRLGTEDRETVSARPAAAPPPSMGTP

[0047] SEQ ID NO.2:

[0048] ATTGTTCGTTCCGCGTGTTTACCGTGAACCGGAAGAATCTTGGAAAATCGACCTGGTTCGTGGTAACCCGCTGGGTCAGCTGGTTTCTAACGGTGCTGAAGGTGAAGCTCCGTGGGTTACCCACGTTCCGATCATCATCGACCCGCGTGTTACCGAACCGGTTACCTCTCTGTCTGG TACCACCCTGTGGGGTCACATGAACATCGGTAACCCGCACTGGCGTGCTCTGGGTCCGGCTACCCCGGTTGCTGTTACCTTCTCTGGTCCGCACGCTTACGTTTCTCCGACCGTTTACGAAACCCGTCCGGCTGCTCCGACCTGGAACTTCACCGCTGTTCACATCGCTGGTGTTC TGCGTAAAGTTGACTCTACCGACGAAACCCTGGCTACCGTTCAGGAAACCGTTCGTGCTTACGAACGTGAATTtGGTGCTGACTGGTCTATGACCGAATCTATCGAATACTTCCGTCGTATCCTGCCGGGTGTTGGTGCTTTCCGTATCGCTATCTCTCTGGCTGACGGTATGTTC AAACTGTCTCAGGAACAGCCGCCGCACGTTCGTGAACGTGTTCGTGCTTCTTCCGCTTGCGAAGCgTCTACCGCTCACCGTGAAGTTGCTGCTCTGATGGGTCGTCTGGGTACCGAAGACCGTGAAACCGTTTCTGCTCGTCCCGGCTGCTGCTCCGCCGCCGTCTATGGGTACCCG

[0049] Using Nde I and Xho I restriction sites, the target gene pai2 was cloned into the vector pET22b using seamless cloning primers PAI2-SC-F (SEQ ID NO.3) and PAI2-SC-R (SEQ ID NO.4), and a His tag was added to obtain the recombinant plasmid pET22b-HTPAI2. To improve the expression of the PAI2 enzyme, pET22b-HTPAI2, together with the molecular chaperone plasmid pG-KJE8, was transformed into competent cells of Escherichia coli BL21(DE3) using the heat shock method. The resulting single colony of the recombinant strain is the engineered strain.

[0050] SEQ ID NO.3: TAAGAAGGAGATATACATATGATGTTCGTTCCGCGTGTTTAC

[0051] SEQ ID NO.4: GTGGTGGTGGTGGTGCTCGAGCGGGGTACCCATAGACGGC

[0052] See the schematic diagram of the pET22b-HTPAI2 recombinant plasmid structure. Figure 1 The SDS-PAGE analysis results of PAI2 co-expressed with the molecular chaperone are shown in the figure below. Figure 2 .

[0053] Example 2: Preparation of the bifunctional enzyme PAI2

[0054] (1) Obtaining crude PAI2 enzyme solution

[0055] The single colony obtained in Example 1 was inoculated into 20 mL of LB medium (containing 50 μg / ml Amp, 25 μg / ml Cm) and cultured at 37°C with shaking for 7-8 h to obtain seed culture. The obtained seed culture was then inoculated into 250 mL of LB medium containing the corresponding antibiotic at an inoculation rate of 1% and cultured at 37°C with shaking until OD. 600 Once the concentration reaches 0.6, add 125 μL IPTG (1M), 2.5 mL inducer 1, and 5 μL inducer 2. Induce at 16℃ for 20 h and collect the bacterial culture. Centrifuge at 8000 rpm and 4℃ for 5 min to collect the cells. Resuspend the cells in lysis buffer (300 mM NaCl, 50 mM Tris-HCl, 10 mM imidazole, pH 8.0), autoclave, and centrifuge to recover the supernatant (4℃, 12000 rpm, 40 min) to obtain the crude PAI2 enzyme solution.

[0056] (2) Purification of crude PAI2 enzyme solution

[0057] His-labeled proteins were separated using Ni-NTA resin. The proteins were washed with washing buffer (300 mM NaCl, 50 mM Tris-HCl, 50 mM imidazole, pH 8.0), then eluted with elution buffer (300 mM NaCl, 50 mM Tris-HCl, 250 mM imidazole, pH 8.0). The proteins were dialyzed three times with storage buffer (300 mM NaCl, 50 mM Tris-HCl, pH 8.0) to remove imidazole from the buffer. Heme chloride was added to bring the heme to enzyme molar ratio to 2:1; after incubation for 12 h, the proteins were dialyzed three times to remove excess heme. The purified protein fraction was analyzed by SDS-PAGE.

[0058] Following the steps outlined in this embodiment, purified enzyme PAI2 was successfully obtained.

[0059] Example 3: Application of the bifunctional enzyme PAI2 in the production of azaproline and proline

[0060] Add 0.116 U PAI2 enzyme and 1 mM LN per milliliter of the reaction mixture. 4 -OH-diaminobutyric acid, 1mM 2-amino-5-hydroxyvalerate, buffer (300mM NaCl, 50mM Tris-HCl, pH 8.0) were added and incubated at 26°C for 1h.

[0061] After the reaction, pre-column derivatization with Fmoc-Cl (9-fluorenylmethoxycarbonyl chloride) was performed: 2 mL of acetonitrile was added, and the mixture was cooled at -20 °C for 10 min, followed by centrifugation at 12000 rpm for 5 min. 1.5 mL of the supernatant was collected, and 100 μL of borax-borate buffer and 200 μL of Fmoc-Cl (20 mM dissolved in acetonitrile) were added, and the mixture was stirred for 5 min. 200 μL of 1-adamantaneamine (0.1 M) was added, and the mixture was incubated for 10 min. The solution was filtered through a membrane and analyzed by LC-MS.

[0062] LC-MS detection:

[0063] LC-MS analysis was performed on a Bruker U3000 liquid chromatography-QTOF tandem mass spectrometer using an Agilent SB-AQ (5 μm, 4.6 mm × 250 mm) column. Samples were eluted at 0.2 mL / min using water and acetonitrile ((v / v): 80:20–30:70, 0–10 min; 30:70–0:100, 10–12 min; 0:100, 12–15 min; 0:100–80:20, 15–16 min; 80:20, 16–25 min), both containing 0.1% (v / v) formic acid.

[0064] According to the operational steps in this embodiment, the biocatalyst PAI2 was successfully used to catalyze the synthesis of azaproline from L-N4-OH-diaminobutyric acid (see...). Figure 4 ), catalyzing the synthesis of proline (Pro) from 2-amino-5-hydroxyvalerate (see) Figure 3 This study demonstrated that PAI2 has multiple functions, including N-N bond synthesis and CN-N bond synthesis, and achieved the simultaneous synthesis of azaproline and proline.

[0065] Example 4: Application of the bifunctional enzyme PAI2 in the production of azaproline and proline

[0066] Add 0.879 U PAI2 enzyme and 10 mM LN per milliliter of the reaction mixture. 4-OH-diaminobutyric acid, 10mM 2-amino-5-hydroxyvalerate, buffer (300mM NaCl, 50mM Tris-HCl, pH 8.0) were added and incubated at 37°C for 6 hours.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. SEQUENCE LISTING <110> Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences <120> A bifunctional enzyme PAI2 with C-N and N-N bond synthesis capabilities, its preparation method and applications <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 235 <212> PRT <213> Streptomyces sp. YIM 121038 <400> 1 Met Phe Val Pro Arg Val Tyr Arg Glu Pro Glu Glu Ser Trp Lys Ile 1 5 10 15 Asp Leu Val Arg Gly Asn Pro Leu Gly Gln Leu Val Ser Asn Gly Ala 20 25 30 Glu Gly Glu Ala Pro Trp Val Thr His Val Pro Ile Ile Ile Asp Pro 35 40 45 Arg Val Thr Glu Pro Val Thr Ser Leu Ser Gly Thr Thr Leu Trp Gly 50 55 60 His Met Asn Ile Gly Asn Pro His Trp Arg Ala Leu Gly Pro Ala Thr 65 70 75 80 Pro Val Ala Val Thr Phe Ser Gly Pro His Ala Tyr Val Ser Pro Thr 85 90 95 Val Tyr Glu Thr Arg Pro Ala Ala Pro Thr Trp Asn Phe Thr Ala Val 100 105 110 His Ile Ala Gly Val Leu Arg Lys Val Asp Ser Thr Asp Glu Thr Leu 115 120 125 Ala Thr Val Gln Glu Thr Val Arg Ala Tyr Glu Arg Glu Phe Gly Ala 130 135 140 Asp Trp Ser Met Thr Glu Ser Ile Glu Tyr Phe Arg Arg Ile Leu Pro 145 150 155 160 Gly Val Gly Ala Phe Arg Ile Ala Ile Ser Leu Ala Asp Gly Met Phe 165 170 175 Lys Leu Ser Gln Glu Gln Pro Pro His Val Arg Glu Arg Val Arg Ala 180 185 190 Ser Phe Ala Cys Glu Ala Ser Thr Ala His Arg Glu Val Ala Ala Leu 195 200 205 Met Gly Arg Leu Gly Thr Glu Asp Arg Glu Thr Val Ser Ala Arg Pro 210 215 220 Ala Ala Ala Pro Pro Pro Ser Met Gly Thr Pro 225 230 235 <210> 2 <211> 705 <212> DNA <213> Synthetic <400> 2 atgttcgttc cgcgtgttta ccgtgaaccg gaagaatctt ggaaaatcga cctggttcgt 60 ggtaacccgc tgggtcagct ggtttctaac ggtgctgaag gtgaagctcc gtgggttacc 120 cacgttccga tcatcatcga cccgcgtgtt accgaaccgg ttacctctct gtctggtacc 180 accctgtggg gtcacatgaa catcggtaac ccgcactggc gtgctctggg tccggctacc 240 ccggttgctg ttaccttctc tggtccgcac gcttacgttt ctccgaccgt ttacgaaacc 300 cgtccggctg ctccgacctg gaacttcacc gctgttcaca tcgctggtgt tctgcgtaaa 360 gttgactcta ccgacgaaac cctggctacc gttcaggaaa ccgttcgtgc ttacgaacgt 420 gaatttggtg ctgactggtc tatgaccgaa tctatcgaat acttccgtcg tatcctgccg 480 ggtgttggtg ctttccgtat cgctatctct ctggctgacg gtatgttcaa actgtctcag 540 gaacagccgc cgcacgttcg tgaacgtgtt cgtgcttctt tcgcttgcga agcgtctacc 600 gctcaccgtg aagttgctgc tctgatgggt cgtctgggta ccgaagaccg tgaaaccgtt 660 tctgctcgtc cggctgctgc tccgccgccg tctatgggta ccccg 705 <210> 3 <211> 42 <212> DNA <213> Synthetic <400> 3 taagaaggag atatacatat gatgttcgtt ccgcgtgttt ac 42 <210> 4 <211> 40 <212> DNA <213> Synthetic <400> 4 gtggtggtgg tggtgctcga gcggggtacc catagacggc 40

Claims

1. A method for preparing a bifunctional enzyme PAI2 with the ability to synthesize CN and NN bonds, characterized in that, Specifically, the steps include the following: (1) Obtaining crude enzyme solution: 125 μL of 1 M IPTG, 2.5 mL of inducer 1, and 5 μL of inducer 2 were added to the culture of engineered bacteria overexpressing the bifunctional enzyme PAI2. The culture was induced at 16℃ for 20 h, centrifuged, and the cells were collected. The cells were then resuspended in lysis buffer, lysed under high pressure, and the supernatant was collected after centrifugation to obtain the crude enzyme solution of PAI2. The composition of inducer 1 was 0.1 M 5-aminolevulinate, 0.1 M ferrous ammonium sulfate, 50 mg / mL arabinose, and the remainder was water. The composition of inducer 2 was 0.25 mg / mL tetracycline, and the remainder was water. The amino acid sequence of the bifunctional enzyme PAI2 is shown in SEQ ID NO.

1. The construction method of the engineered bacteria overexpressing the bifunctional enzyme PAI2 was to use the Nde I and Xho I restriction sites to express the target gene. pai2 The plasmid pET22b was cloned into the vector pET22b and a His tag was added to obtain the recombinant plasmid pET22b-HTPAI2. pET22b-HTPAI2, along with the molecular chaperone plasmid pG-KJE8, was transformed into competent cells of *E. coli* BL21(DE3) using a heat shock method. A single colony of the recombinant strain was obtained, which is the engineered bacterium. The target gene... pai2 The nucleotide sequence is as shown in SEQ ID NO.2; (2) Purification of crude enzyme solution: His-labeled protein was separated by Ni-NTA resin. After washing the protein with washing buffer, the protein was eluted with elution buffer. Then, the protein was dialyzed three times with storage buffer to remove imidazole in the buffer. Heme chloride was added to make the ratio of heme to enzyme 2:

1. After incubation for 12 h, the protein was dialyzed three times to remove excess heme.

2. The preparation method according to claim 1, characterized in that, The preparation method of the engineering bacteria liquid in step (1) is as follows: the engineering bacteria is inoculated into LB culture medium containing antibiotics to obtain seed liquid, the seed liquid is inoculated into LB culture medium containing antibiotics with 1% inoculation amount, and the culture is carried out until OD 600 is 0.

6.

3. The preparation method according to claim 1, characterized in that, The washing buffer in step (2) consists of 300 mM NaCl, 50 mM Tris-HCl, 50 mM imidazole, with the remainder being water, and pH 8.0; the elution buffer consists of 300 mM NaCl, 50 mM Tris-HCl, 250 mM imidazole, with the remainder being water, and pH 8.0; the storage buffer consists of 300 mM NaCl, 50 mM Tris-HCl, with the remainder being water, and pH 8.

0.

4. The application of the bifunctional enzyme PAI2 with CN-N bond and NN-N bond synthesis capabilities obtained by any of the preparation methods described in claims 1-3, characterized in that, L-N 4 -OH- diaminobutyric acid and 2-amino-5-hydroxyvaleric acid as substrates, adding a bifunctional enzyme PAI2 with C-N bond and N-N bond synthesis ability to catalyze to obtain azetidine and proline.

5. The application according to claim 4, characterized in that, The LN 4 The concentrations of -OH-diaminobutyric acid and 2-amino-5-hydroxyvalerate are both 1-10 mM, and the amount of the bifunctional enzyme PAI2 with CN and NN bond synthesis capabilities added is 0.087 U / g substrate to 0.879 U / g substrate; the catalysis is carried out at 26℃-37℃ for 1-6 h.

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