阅读说明：本技术 一种合成(s)-6-硝基正亮氨酸的方法及其催化酶 (Method for synthesizing (S) -6-nitro norleucine and catalytic enzyme thereof ) 是由 钟冠男 卞小莹 张友明 庞琳琳 于 2021-08-13 设计创作，主要内容包括：本发明属于化合物合成技术领域,具体涉及由生物酶催化的化合物合成方法及催化该化学合成的酶。一种合成(S)-6-硝基正亮氨酸的方法,其合成方程式为：本发明提供的合成(S)-6-硝基正亮氨酸的方法使用生物酶作为催化剂,以天然氨基酸为原料,一步生成目标化合物,缩减反应步骤；该反应使用水作为反应溶剂,避免有机溶剂的使用,后处理过程相对比较简单,酶催化剂可以通过生物降解,对环境污染小；避免使用有毒有害、高度活泼的化学试剂,在中性pH值和室温下即可发生反应,避免了有机化学反应中苛刻的反应条件,因此更加安全。(The invention belongs to the technical field of compound synthesis, and particularly relates to a compound synthesis method catalyzed by a biological enzyme and an enzyme for catalyzing the chemical synthesis. A method for synthesizing (S) -6-nitro-norleucine has a synthesis equation: the method for synthesizing (S) -6-nitro-norleucine uses biological enzyme as a catalyst, takes natural amino acid as a raw material, generates a target compound in one step, and reduces reaction steps; the reaction uses water as a reaction solvent, avoids the use of an organic solvent, has relatively simple post-treatment process, can degrade an enzyme catalyst by biology, and has little pollution to the environment; avoids the use of toxic, harmful and highly active chemical reagents, can react at neutral pH value and room temperature, avoids harsh reaction conditions in organic chemical reaction, and is more favorableAnd (4) safety.)

1. A method for synthesizing (S) -6-nitro-norleucine is characterized in that the synthesis equation is as follows:

wherein, in the formula (1), R₁、R₂、R₃Is H, X is S or CH₂Or alternatively;

R₁、R₂is H, R₃Is OH, X is CH₂Or alternatively;

R₁is CH₃，R₂、R₃Is H, X is CH₂Or alternatively;

R₁、R₃is H, R₂Is Ac, X is CH₂；

The chiral configuration of the carbon atom is S;

the synthesis process of the compound shown in the formula (2) is as follows: carrying out oxidation-reduction reaction on the compound shown in the formula (1) under the action of catalytic enzyme to generate a compound shown in a formula (2);

the ratio of the compound shown in the formula (1) to the catalytic enzyme is 5:1 to 50: 1.

2. The method for synthesizing (S) -6-nitronorleucine according to claim 1, wherein the catalytic enzyme is an enzyme comprising the amino acid sequence:

QEXXXDXXFDDXXXXXXXGXXXXXKXEXXXNXXDEMGNGXXXXXHXXXF；

GXXXXTEXXXPXR(X)_17-21YHXXHXXXDXXHXXXXXXNVXXP(X)_12-16GXXXRXNXSXXYLD；

wherein X represents any amino acid and the subscript numbers represent the range of the number of any amino acid.

3. The method for synthesizing (S) -6-nitronorleucine according to claim 1, wherein phenazine methosulfate as an electron mediator is added to the reaction system at a final concentration of 20 to 50. mu.M.

4. The process for synthesizing (S) -6-nitronorleucine according to claim 1, wherein the solvent in the reaction system is water.

5. A catalytic enzyme catalysing the process of any one of claims 1 to 4, wherein the catalytic enzyme comprises the amino acid sequence:

QEXXXDXXFDDXXXXXXXGXXXXXKXEXXXNXXDEMGNGXXXXXHXXXF；

GXXXXTEXXXPXR(X)_17-21YHXXHXXXDXXHXXXXXXNVXXP(X)_12-16GXXXRXNXSXXYLD；

wherein X represents any amino acid and the subscript numbers represent the range of the number of any amino acid.

6. The catalytic enzyme of claim 5, wherein the catalytic enzyme is BelK or Hrmi.

Technical Field

The invention belongs to the technical field of compound synthesis, and particularly relates to a compound synthesis method catalyzed by a biological enzyme and an enzyme for catalyzing the compound synthesis.

Background

Arginase can hydrolyze L-arginine in human body into L-ornithine and urea, NO synthetase can catalyze the oxidation of L-arginine to generate L-citrulline and NO, both of the L-arginine and the NO synthetase take L-arginine as a substrate and have strong substrate competitiveness, so that the metabolism of the NO synthetase on the L-arginine can be promoted by inhibiting the activity of the arginase, the NO concentration in the human body can be increased, and the L-arginine hydrolase inhibitor can be used for treating diseases caused by NO deficiency, such as male sexual dysfunction, asthma, atherosclerosis and the like. Research shows that (S) -6-nitro norleucine has strong inhibition effect on arginase, and dissociation constant kd of the two is 60 mu M (J.am.chem.Soc.2008,130, 17254-17255). Therefore, (S) -6-nitro norleucine has larger drug potential.

Currently, only one example of chemical synthesis of (S) -6-nitronorleucine is reported (Eur.J.org.chem.2006, 1525-1534). The method takes L-lysine with diamino protecting group as raw material, and comprises 4 steps of carboxyl protection, 6-amino deprotection, 6-amino oxidation to nitro, amino removal and carboxyl protecting group removal in sequence to generate (S) -6-nitro-norleucine, and the total yield is about 48%. The chemical synthesis of the compound has various disadvantages, such as multi-step protection and deprotection of amino and carboxyl, complicated reaction process and low atom economy; the reaction process comprises hydrogenation reaction and heating reflux reaction, and the reaction conditions are harsh and have high risk; various organic solvents, strong acid and strong base are used in the reaction and post-treatment processes, so that the environment pollution is easily caused, and the concept of green chemistry is not met; the total reaction yield is low, the production cost is high, and the economic benefit is poor.

Disclosure of Invention

The invention aims to make up the defects of the existing chemical synthesis of (S) -6-nitronorleucine and provide a novel biosynthesis method for synthesizing (S) -6-nitronorleucine. The method uses biological enzyme as a catalyst, uses natural amino acid as a raw material, generates (S) -6-nitro-norleucine in one step, avoids the use of chemical reagents and harsh reaction conditions, does not need to introduce and remove protective groups, has high reaction efficiency, meets the requirements of green chemistry, and is safe and nontoxic.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for synthesizing (S) -6-nitro-norleucine has a synthesis equation:

wherein, in the formula (1), R₁、R₂、R₃Is H, X is S or CH₂Or alternatively;

R₁、R₂is H, R₃Is OH, X is CH₂Or alternatively;

R₁is CH₃，R₂、R₃Is H, X is CH₂Or alternatively;

R₁、R₃is H, R₂Is Ac, X is CH₂；

The chiral configuration of the carbon atom is S;

the synthesis process of the compound shown in the formula (2) is as follows: the compound shown in the formula (1) generates oxidation-reduction reaction under the action of catalytic enzyme to generate the compound shown in the formula (2).

In a preferred embodiment of the present invention, in the formula (1), the catalytic enzyme is a biological enzyme, and the biological enzyme is an enzyme comprising the following amino acid sequence:

QEXXXDXXFDDXXXXXXXGXXXXXKXEXXXNXXDEMGNGXXXXXHXXXF；

GXXXXTEXXXPXR(X)_17-21YHXXHXXXDXXHXXXXXXNVXXP(X)_12-16GXXXRXNXSXXYLD；

wherein X represents any amino acid and the subscript numbers represent the range of the number of any amino acid.

Further preferably, Phenazine Methosulfate (PMS) is added to the reaction system as an electron mediator at a final concentration of 20 to 50. mu.M.

Further preferably, the solvent in the reaction system is water.

The invention also provides a catalytic enzyme, which comprises the following amino acid sequence:

QEXXXDXXFDDXXXXXXXGXXXXXKXEXXXNXXDEMGNGXXXXXHXXXF；

GXXXXTEXXXPXR(X)_17-21YHXXHXXXDXXHXXXXXXNVXXP(X)_12-16GXXXRXNXSXXYLD；

wherein X represents any amino acid and the subscript numbers represent the range of the number of any amino acid.

Further preferably, the catalytic enzyme is BelK or HrmI.

Compared with the existing chemical synthesis method, the method for synthesizing (S) -6-nitro-norleucine uses biological enzyme as a catalyst, takes natural amino acid as a raw material, generates a target compound in one step, and reduces reaction steps; the reaction uses water as a reaction solvent, avoids the use of an organic solvent, has relatively simple post-treatment process, can degrade an enzyme catalyst by biology, and has little pollution to the environment; toxic, harmful and highly active chemical reagents are avoided from being used, the reaction can be carried out at neutral pH value and room temperature, and harsh reaction conditions in organic chemical reaction are avoided, so that the method is safer; the enzyme catalyst is obtained by a protein heterologous expression method, is cheap and easy to obtain, and can be prepared in large quantities; the high selectivity and high efficiency of the enzyme catalysis reaction avoid the introduction and the removal of protecting groups, reduce the occurrence of side reactions, introduce the atoms in reactants into products as much as possible and realize the maximization of atom economy.

Drawings

FIG. 1 is a SDS-PAGE of HrmI and BelK prepared in the examples of the present invention and purified site-directed muteins of BelK; in the figure, 1: hrmi (41.33kDa), 2: BelK (42.38kDa), 3: BelK E205A, 4: BelK H215A, 5: BelK E269A, 6: BelK H299A, 7: BelK D303A, 8: BelK H306A, 9: BelK Y295F;

FIG. 2 is a HPLC-ESI-MS graph showing that BelK and Hrmi catalyze L-lysine to produce (S) -6-nitro-norleucine;

FIG. 3 is a HPLC-ESI-MS graph of BelK catalyzing L-lysine methyl ester and N α -acetyl-lysine to (S) -6-nitro-norleucine;

FIG. 4 is a HPLC-ESI-MS graph of BelK catalyzing 5-hydroxy-L-lysine to (S) -5-hydroxy-6-nitro-norleucine;

FIG. 5 is a HPLC-ESI-MS diagram of BelK catalyzing the formation of S- (2-nitroethyl) -L-cysteine from S- (2-aminoethyl) -L-cysteine;

FIG. 6 shows the results of the detection of catalytic activity of each site-directed mutant protein of BelK.

Detailed Description

In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The primer synthesis and gene sequencing of the catalytic enzyme involved in the embodiment of the invention are completed by Shanghai biological engineering Co., Ltd; the bacterial plasmid small-extraction kit is purchased from Tiangen Biotechnology, Inc.; the PCR reaction system 2 × Ape × HF FS PCR Master Mix was purchased from Aikery bioengineering, Inc.; the nickel column packing was purchased from Shanghai Bogelong Biotechnology Ltd; the polyacrylamide gel electrophoresis (SDS-PAGE) pre-gel used was purchased from Kinry Biotech, Inc.; the Protein Marker is Transgene Blue Plus IV Protein Marker which is purchased from Beijing all-purpose gold biotechnology (TransGen Biotech) limited; SDS-PAGE sample buffer was purchased from Beijing ancient China Changsheng Biotechnology Co., Ltd; SDS-PAGE fast staining solution was purchased from Beijing Rui Boxing Biotech, Inc.; protein ultrafiltration centrifuge tubes were purchased from Merck Millipore; protein desalting column PD-10 was purchased from GE life sciences, USA; HPLC was purchased from Fisher Chemical, USA, using acetonitrile and methanol.

Example 1, this example used BelK as a catalytic enzyme to synthesize (S) -6-nitro-norleucine by the following steps:

1. synthesis of BelK catalytic enzyme

(1) Gene synthesis and electrotransformation

The applicant entrusts Suzhou Jinzhi biotechnology limited to perform codon optimization and gene synthesis on gene belK (protein _ id ═ ARO49579.1), and the gene belK is connected to pET28a (+) expression vector through NdeI + HindIII enzyme cutting sites to obtain pET28a + belK protein expression plasmid. Escherichia coli BL21(DE3) was subjected to shake cultivation at 37 ℃ for 5 hours in a 1.5mL centrifuge tube, centrifuged at 12000rpm for 1min, the supernatant was discarded, 1mL of sterile water was added thereto, centrifuged at 12000rpm for 1min and washed twice, the supernatant was discarded, 1. mu.L of plasmid solution was added thereto, and the mixture was mixed well and transferred to an electric rotor. Placing the electric rotating cup into an electric rotating instrument, setting the voltage to 1350V for electric conversion, quickly adding 1mL of fresh LB culture medium into the electric rotating cup after the electric rotating cup is finished, transferring the electric rotating cup into a 1.5mL centrifuge tube, culturing for 1h in a shaking way at 37 ℃, coating the electric rotating cup on an LB solid plate containing kanamycin (50 mu g/mL), and performing inversion culture at 37 ℃ overnight.

The sequence of the belK gene after codon optimization of escherichia coli is as follows:

ATGACCACCGAGCGTAATCTGGATATGCCGGACCCGCAGCGCAACGATTTTCCGCTGCTGGCCGAATTTACAGCCGCCCTGACCAGTGTTAGCTTTGGCACCTGGCAGGAGCTGGTGGATGACTATGCCCATCGCGCAGAACTGGCAGGCGAATGTCGCCGCCTGGCCGATCTGGCATTTCGTGGTCGCGATGCCGCAGCCCGTGAACACCTGCATGACATCCTGACCGTGGTGTACGCATACGAATTTAGCCAGAGCGCAGCCCGTAATCCGGATCAGGATCCTCAGCCGATCCTGCGTGACGTGACCAGCGTGCTGGAGAACGCCATGCTGGATAATGAATTTCGCCAGGTGCCGGAAGAACTGTTAACCGGCTATCCGAGCGGTGAGAAAGAATATGTGCGCTGGCTGAAAGCCCTGATCCAGGATCATCCGGCAAGCGCACATCCGATGTATGCCGAGCACCTGACAAACGCAGCAACCGTGGAAGATATCCGCTTTCTGCTGGCACAGGAAACAAGCCTGGATCCTCGCTTCGACGATATCCTGGCCGTTATGCAGTTTGGCGCCACAGGCGCCGAGAAGATGGAGATTGCCGCAAACTACTGGGACGAAATGGGCAATGGCGAGTTTGCAGATGTGCATACCACCCTGTTTAGCCAGTGCCTGACCAGCATCGGCGTTGATCGTGGCTACATCGAAAGCAATCTGCTGCTGGCCGCCAAAGAGTGCGGCAATATTAGTGCAGGCCTGGCCCTGAGCCGCCGTCATTATCTGCGTGCCATCGGCTATTACGGCGTGACCGAATTTCTGGCACCGCGCCGCTTTCGTCAGCTGGTTACAGCCTGGGATCGCTTAGGTCTGCCCCCTGAAGGTAAGGTGTACCACGACATCCACATCAACGTGGATGCCCACCATGCCGCCGGTTGGTATAAGAACGTGATTGGCCCGGTTGTTGAACGTGATCCGGCCGCAGGTCGTGAAATTGCCCTGGGCACCTTCGTTCGCCTGAATACCAGCGCCTATTATCTGGATCGCGTGCTGGAAGTGTGCCAGAAACAACCGCTGCCGGCATAA。

the BelK protein sequence is as follows:

MTTERNLDMPDPQRNDFPLLAEFTAALTSVSFGTWQELVDDYAHRAELAGECRRLADLAFRGRDAAAREHLHDILTVVYAYEFSQSAARNPDQDPQPILRDVTSVLENAMLDNEFRQVPEELLTGYPSGEKEYVRWLKALIQDHPASAHPMYAEHLTNAATVEDIRFLLAQETSLDPRFDDILAVMQFGATGAEKMEIAANYWDEMGNGEFADVHTTLFSQCLTSIGVDRGYIESNLLLAAKECGNISAGLALSRRHYLRAIGYYGVTEFLAPRRFRQLVTAWDRLGLPPEGKVYHDIHINVDAHHAAGWYKNVIGPVVERDPAAGREIALGTFVRLNTSAYYLDRVLEVCQKQPLPA。

(2) protein expression and purification

Electrically transforming plasmid pET28a + belK into Escherichia coli BL21(DE3), selecting single colony, inoculating to 50mL LB (containing kanamycin 50 ug/mL), placing into a shaker at 37 deg.C and 200rpm for 10h, transferring to 2L LB (containing kanamycin 50 ug/mL) after gene sequencing is correct, placing into a shaker at 37 deg.C and 200rpm for 4h to obtain bacterial solution OD₆₀₀When the concentration is about 0.6, ferric chloride hexahydrate solution is added to the final concentration of 0.2mM, inducer Isopropyl-beta-D-thiogalactoside (IPTG) is added to the final concentration of 0.1mM, the mixture is placed in a shaker at 25 ℃ and 200rpm for 24 hours, and then the thalli are centrifugally collected. Adding bacteria-breaking buffer solution (K)₂HPO₄The bacterial cells were resuspended in 50mM NaCl 300mM, imidazole 5mM, and glycerol 10% (v/v) and adjusted to pH 7.5 with hydrochloric acid, and 5mL of the lysis buffer solution corresponded to 1g of the bacterial cells. The bacteria are broken three times by using an ultrahigh pressure cell breaker (about 700 bar) until the liquid is semitransparent, and the liquid is centrifuged for 1h at 14000rpm and 4 ℃. After centrifugation, the supernatant was applied to a nickel column, and proteins were eluted sequentially with 10mM, 25mM, 50mM, and 500mM imidazole buffers, and the eluates were stored at 4 ℃ or in an ice bath. Each concentration was checked by SDS-PAGE. Collecting target protein according to electrophoresis result, concentrating protein with 10kDa protein ultrafiltration centrifugal tube, removing imidazole with protein desalting column PD-10, and exchanging protein purification solution with desalting buffer solution (K)₂HPO₄50mM, NaCl 100mM, glycerol 10% (v/v), DTT 1 mM). Protein concentration was measured using a microplate reader. Packaging into a tube per 20 μ L, quickly freezing in liquid nitrogen, and storing at-80 deg.C. The protein yield was about 65 mg/L. The electrophoresis results are shown in FIG. 1.

2. BelK catalyzes the production of (S) -6-nitronorleucine

TABLE 1 reaction system (Total volume 100. mu.L)

According toSequential addition of H in the order of Table 1₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 hr.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 12.4min, molecular weight [ M + H ]]⁺410, this peak is absent in the absence of L-lysine, NADH, PMS or BelK (fig. 2), and high resolution mass spectrometry shows that the peak has an exact molecular weight of [ M + H []⁺410.1385, the molecular formula is presumed to be C₁₈H₂₄N₃O₆S⁺(calculated value: [ M + H ]]⁺410.1380), consistent with (S) -6-nitro-norleucine after DNSC derivation, BelK is presumed to oxidize the 6-amino group of L-lysine to (S) -6-nitro-norleucine.

To further confirm that the product is (S) -6-nitronorleucine, applicants chemically synthesized (S) -6-nitronorleucine standard according to the literature method (Eur.J.org.chem.2006,1525-1534), derivatized (S) -6-nitronorleucine with DNSC according to the above method, and HPLC-ESI-MS showed that (S) -6-nitronorleucine after DNSC derivatization has the same retention time, molecular weight and UV absorption spectrum as the product synthesized by the method of the present invention, as shown in FIG. 2, further showing that BelK catalytic enzyme can oxidize the 6-amino group of L-lysine to (S) -6-nitronorleucine with high regioselectivity, while the 2-amino group is not oxidized at all.

Example 2, this example uses HrmI as a catalytic enzyme to synthesize (S) -6-nitro-norleucine by the following steps:

1. the synthesis of the enzyme is catalyzed by HrmI (protein _ id) ═ AEH 41787.1): the synthesis process and method are the same as BelK in example 1, and the electrophoresis result is shown in FIG. 1.

The hrmI gene E.coli codon optimized sequence is as follows:

ATGATCCCGGAAAGTTTTAAAATCGACCGCAGCGTCGTTGAAGAATTTCTGGCGCTGGATCCGGACGCTTGGGAACGTCTGAACGCAGATTATACCGCACGTCGTCGTATTGGCGAAGCGTGCCGTGCACTGAGTCGTCACGCATTTGTTGAAGAAGATCCGTCTGCACTGGAAGAACTGCACGATGTTCTGGCGCTGATTTATCAGCAGGATTTTTCTGGCGCACCGGTTGAACTGCTGGGTTGCGAAACCCAACCGGTTCTGCGCGATATTGCAGCAATTCTGGAAGGCGCTGTTCTGGCAGCGGAACTGGATAGCATTAGCGAAGAACAGATTAGCGCGTATCCGCGTTCTGGTAAAGAGTACGTCCACTGGCTGAAACGCGTTATTGGCGAACATCCGGCAGCAGGTCATCCGTTTTATCGCGATTTTGTTCCGACCCGCGCAACCGAAGGCGATTTTCGTTTCTACCTGGCACAGGAAACCAACCTGGATCCGAAATTCGACGACATCCTGGCGTTTATGCAAATTGGCGCAGCACCGGACGAAAAAATGGAAATTGCGGGCAACTACTGGGACGAAATGGGTAACGGTAAACCGGCAGAGGTTCACACCGCTATGTTTGCACATGCACTGGACGCACTGGACGTTAACGACGATTATATCCGCCGTAATCTGCTGCCGGAAGCAAAAGCAAGCGGTAATCTGGCAAGCTGTCTGGCGATTAGCCGCCGTCATTACTACAAAAGCGTTGGCTTCTTTGGCGTCACCGAATATCTGGTTCCGCGTCGCTTTAAACTGGTCGTTGATCGTTGGGCTGATATTGGTCTGCCGCGCGAAGGTATTGCGTATCACGACGCGCATATTTCCATTGATGCGGTTCACGCAAGCGGTTGGTTTAAAAACGTTATTGCGCCGGCAGTTGATCGCGATCCGCGCGTTGGTCGCGAAATTGCAGTTGGTGCACTGATTCGTCTGAATAGCAGCCAACGTTACCTGGATTCCCTGCTGATGCATCTGCATCATGATAGCGCAGCACATACCAGTTAA。

the Hrmi protein sequence is as follows:

MIPESFKIDRSVVEEFLALDPDAWERLNADYTARRRIGEACRALSRHAFVEEDPSALEELHDVLALIYQQDFSGAPVELLGCETQPVLRDIAAILEGAVLAAELDSISEEQISAYPRSGKEYVHWLKRVIGEHPAAGHPFYRDFVPTRATEGDFRFYLAQETNLDPKFDDILAFMQIGAAPDEKMEIAGNYWDEMGNGKPAEVHTAMFAHALDALDVNDDYIRRNLLPEAKASGNLASCLAISRRHYYKSVGFFGVTEYLVPRRFKLVVDRWADIGLPREGIAYHDAHISIDAVHASGWFKNVIAPAVDRDPRVGREIAVGALIRLNSSQRYLDSLLMHLHHDSAAHTS。

2. hrmi catalyzes the production of (S) -6-nitronorleucine

TABLE 2 reaction system (Total volume 100. mu.L)

H was added in the order of Table 2₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, Hrmi protein and NADH are added finally, the mixture is mixed evenly and the reaction is started, and the mixture is placed for 2 hours at the temperature of 28 ℃.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 12.4min, molecular weight [ M + H ]]⁺410, this peak is absent in the absence of L-lysine, NADH, PMS or HrmI (fig. 2), and high resolution mass spectrometry shows that the peak has an exact molecular weight of [ M + H []⁺410.1384, the molecular formula is presumed to be C₁₈H₂₄N₃O₆S⁺(calculated value: [ M + H ]]⁺410.1380), corresponding to (S) -6-nitro-norleucine after DNSC derivation, Hrmi is presumed to oxidize the 6-amino group of L-lysine to produce (S) -6-nitro-norleucine.

To further confirm that the product is (S) -6-nitronorleucine, applicants chemically synthesized (S) -6-nitronorleucine standard according to the literature method (Eur.J.org.chem.2006,1525-1534), derivatized (S) -6-nitronorleucine with DNSC according to the above method, and HPLC-ESI-MS showed that (S) -6-nitronorleucine after DNSC derivatization has the same retention time, molecular weight and UV absorption spectrum as the product synthesized by the method of the present invention, as shown in FIG. 2, further demonstrated that the Hrmi catalytic enzyme can oxidize the 6-amino group of L-lysine to (S) -6-nitronorleucine with high regioselectivity, while the 2-amino group is not oxidized at all.

Example 3, this example used BelK as a catalytic enzyme, and L-lysine methyl ester as a substrate to synthesize (S) -6-nitro-norleucine by the following steps:

1. synthesis of BelK catalytic enzyme: the synthesis process and method are the same as BelK in example 1, and the electrophoresis result is shown in FIG. 1.

2. BelK catalyzes L-lysine methyl ester to generate (S) -6-nitro-norleucine

TABLE 3 reaction system (Total volume 100. mu.L)

H was added in the order of Table 3₂O、K₂HPO₄Buffer solution, L-lysine methyl ester, phenazine methyl sulfate, BelK protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 hr.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 12.4min, molecular weight [ M + H ]]⁺410, this peak is absent in the absence of BelK (fig. 3), and high resolution mass spectrometry shows this peakThe precise molecular weight is [ M + H ]]⁺410.1385, the molecular formula is presumed to be C₁₈H₂₄N₃O₆S⁺(calculated value: [ M + H ]]⁺410.1380) corresponding to (S) -6-nitro-norleucine after DNSC derivatization. In addition, the retention time, molecular weight and UV absorption spectrum of this peak were all the same as DNS- (S) -6-nitro-norleucine produced by catalyzing L-lysine production by BelK. HPLC-ESI- (HR) MS did not detect the molecular weight of DNS- (S) -6-nitronorleucine methyl ester (C)₁₉H₂₆N₃O₆S⁺Calculating the value: [ M + H ]]⁺424.1537) indicating that in the catalytic process BelK could remove the methyl protecting group from the L-lysine carboxy group to produce (S) -6-nitro-norleucine and no (S) -6-nitro-norleucine methyl ester.

Example 4, this example uses BelK as catalytic enzyme, and N alpha-acetyl protected L-lysine as substrate, synthesis of (S) -6-nitro-N-leucine, the specific steps are as follows:

1. synthesis of BelK catalytic enzyme: the synthesis process and method are the same as BelK in example 1, and the electrophoresis result is shown in FIG. 1.

2. BelK catalyzes N alpha-acetyl-L-lysine to generate (S) -6-nitro norleucine

TABLE 4 reaction system (Total volume 100. mu.L)

H was added in the order of Table 4₂O、K₂HPO₄Buffer solution, N alpha-acetyl-L-lysine, phenazine methyl sulfate, BelK protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 hr.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Flow ofMoving phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 12.4min, molecular weight [ M + H ]]⁺410, this peak is absent in the absence of BelK (fig. 3), and high resolution mass spectrometry shows that this peak has an exact molecular weight of [ M + H []⁺410.1383, the molecular formula is presumed to be C₁₈H₂₄N₃O₆S⁺(calculated value: [ M + H ]]⁺410.1380) corresponding to (S) -6-nitro-norleucine after DNSC derivatization. In addition, the retention time, molecular weight and UV absorption spectrum of this peak were all the same as DNS- (S) -6-nitro-norleucine produced by catalyzing L-lysine production by BelK. HPLC-ESI- (HR) MS did not detect the molecular weight of (S) -2-acetyl-6-nitro-norleucine, indicating that BelK can remove the acetyl protecting group of N alpha-acetyl-L-lysine during the catalysis process to produce (S) -6-nitro-norleucine and not (S) -2-acetyl-6-nitro-norleucine.

Example 5, this example used BelK as a catalytic enzyme, and 5-hydroxy-L-lysine as a substrate, to synthesize (S) -5-hydroxy-6-nitro-norleucine, the specific steps were as follows:

1. synthesis of BelK catalytic enzyme: the synthesis process and method are the same as BelK in example 1, and the electrophoresis result is shown in FIG. 1.

2. BelK catalyzes 5-hydroxy-L-lysine to generate (S) -5-hydroxy-6-nitro-norleucine

TABLE 5 reaction system (Total volume 100. mu.L)

H was added in the order of Table 5₂O、K₂HPO₄Buffer solution, 5-hydroxy-L-lysine, phenazine methyl sulfate, BelK protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 hr.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 10.8min, molecular weight [ M + H ]]⁺426, this peak is absent in the absence of BelK (fig. 4), and high resolution mass spectrometry shows that this peak has an exact molecular weight of [ M + H []⁺426.1336, the molecular formula is presumed to be C₁₈H₂₄N₃O₇S⁺(calculated value: [ M + H ]]⁺426.1329) corresponding to (S) -5-hydroxy-6-nitro-norleucine after DNSC derivatization. Thus, BelK can catalyze the oxidation of 5-hydroxy-L-lysine to generate (S) -5-hydroxy-6-nitro-norleucine.

Example 6, this example uses BelK as a catalytic enzyme and S- (2-aminoethyl) -L-cysteine as a substrate to synthesize S- (2-nitroethyl) -L-cysteine by the following steps:

1. synthesis of BelK catalytic enzyme: the synthesis process and method are the same as BelK in example 1, and the electrophoresis result is shown in FIG. 1.

2. BelK catalyzes S- (2-aminoethyl) -L-cysteine to generate S- (2-nitroethyl) -L-cysteine

TABLE 6 reaction system (Total volume 100. mu.L)

H was added in the order of Table 6₂O、K₂HPO₄Buffer solution, S- (2-aminoethyl) -L-cysteine, phenazine methosulfate, BelK protein,and finally adding NADH, mixing uniformly, starting the reaction, and standing at 28 ℃ for 2 h.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a new peak at a retention time of about 12.3min, molecular weight [ M + H ]]⁺428, this peak is absent in the absence of BelK (fig. 5), and high resolution mass spectrometry shows that this peak has an exact molecular weight of [ M + H []⁺428.0936, the molecular formula is presumed to be C₁₇H₂₂N₃O₆S₂ ⁺(calculated value: [ M + H ]]⁺428.0945) corresponding to S- (2-nitroethyl) -L-cysteine after DNSC derivatization. Thus, BelK can catalyze the oxidation of S- (2-aminoethyl) -L-cysteine to produce S- (2-nitroethyl) -L-cysteine.

In order to further and deeply research the functional sites and the catalytic mechanism of the catalytic enzyme provided by the invention, the applicant takes the BelK catalytic enzyme as an example, carries out site-directed mutation on part of amino acids (shown by underlining), and proves that the sites have important effects on the catalytic function of the enzyme, and particularly, see examples 7-14.

QEXXXDXXFDDXXXXXXXGXXXXXKXEXXXNXXDEMGNGXXXXXHXXXF；

GXXXXTEXXXPXR(X)_17-21 YHXXHXXXDXXHXXXXXXNVXXP(X)_12-16GXXXRXNXSXXYLD；

Wherein X represents an arbitrary amino acid, and the number represents the number range of the arbitrary amino acid.

Example 7, this example uses the BelK E205A point mutant protein as catalytic enzyme, uses L-lysine as substrate, tests the influence of E205 amino acid residue on the catalytic activity of BelK protein, and the concrete steps are as follows:

1. synthesis of BelK E205A Point mutant protein

(1) Construction of BelK E205A Point mutation plasmid

Using pET28a + belK plasmid as template and primer BelK-E205A-F (5' -GCAAACTACTGGGAC)GCAATGGGCAATGGCGAG-3 ') and BelK-E205A-R (5' -CTCGCCATTGCCCAT)TGCGTCCCAGTAGTTTGC-3'), and the PCR system is shown in Table 7. After the PCR is finished, 1 mu L of DpnI restriction enzyme is added into the PCR system to remove the template, and then desalting is carried out for 30min by a desalting membrane. Culturing Escherichia coli BL21(DE3) in a 1.5mL centrifuge tube at 37 ℃ for 5h by shaking table, centrifuging at 12000rpm for 1min, discarding the supernatant, adding 1mL sterile water at 12000rpm, centrifuging for 1min, washing twice, discarding the supernatant, adding all PCR systems, mixing, and transferring to an electric rotating cup. Placing the electric rotating cup into an electric rotating instrument, setting the voltage to 1350V for electric conversion, quickly adding 1mL of fresh LB culture medium into the electric rotating cup after the electric rotating cup is finished, transferring the electric rotating cup into a 1.5mL centrifuge tube, culturing for 1h in a shaking way at 37 ℃, coating the electric rotating cup on an LB solid plate containing kanamycin (50 mu g/mL), and performing inversion culture at 37 ℃ overnight. Single colonies were picked, inoculated into 1mL LB (containing 50. mu.g/mL kanamycin), placed on a shaker at 37 ℃ and 960rpm for 5h, and sequenced.

TABLE 7 PCR reaction System (Total volume 20. mu.L)

(2) Expression and purification of BelK E205A point mutant protein

Selecting correctly sequenced BL21(DE3), pET28a + belK E205A, transferring to 2L LB (containing 50 ug/mL kanamycin), placing into a shaker at 37 deg.C and 200rpm for 4h, and waiting for bacterial liquid OD₆₀₀When the concentration is about 0.6, ferric chloride hexahydrate solution is added to the final concentration of 0.2mM, and inducer Isopropyl-beta-D-thiogalactoside (Isopropypyl-beta-D-thiogal) is addedactinopyranoside, IPTG) to a final concentration of 0.1mM, shaking at 200rpm for 24h at 25 ℃ and then centrifuging to collect the cells. Adding bacteria-breaking buffer solution (K)₂HPO₄The bacterial cells were resuspended in 50mM NaCl 300mM, imidazole 5mM, and glycerol 10% (v/v) and adjusted to pH 7.5 with hydrochloric acid, and 5mL of the lysis buffer solution corresponded to 1g of the bacterial cells. The bacteria are broken three times by using an ultrahigh pressure cell breaker (about 700 bar) until the liquid is semitransparent, and the liquid is centrifuged for 1h at 14000rpm and 4 ℃. After centrifugation, the supernatant was applied to a nickel column, and proteins were eluted sequentially with 10mM, 25mM, 50mM, and 500mM imidazole buffers, and the eluates were stored at 4 ℃ or in an ice bath, and each concentration was checked by SDS-PAGE. Collecting target protein according to electrophoresis result, concentrating protein with 10kDa protein ultrafiltration centrifugal tube, removing imidazole with protein desalting column PD-10, and exchanging protein purification solution with desalting buffer solution (K)₂HPO₄50mM, NaCl 100mM, glycerol 10% (v/v), DTT 1 mM). Protein concentration was measured using a microplate reader. Packaging into a tube per 20 μ L, quickly freezing in liquid nitrogen, and storing at-80 deg.C. The electrophoresis results are shown in FIG. 1.

2. Living test line of BelK E205A

TABLE 8 reaction system (Total volume 100. mu.L)

H was added in the order of Table 8₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK E205A protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 h.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t ═0min,5％B；T＝3min,5％B；T＝18min,95％B；T＝22min,95％B；T＝23min,5％B；and T＝25min,5％B。

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK E205A assay in vivo lines, molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (fig. 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of E205 to alanine, and that the E205 amino acid residue had a significant effect on the catalytic function of BelK.

Example 8, this example uses the BelK H215A point mutant protein as a catalytic enzyme, and uses L-lysine as a substrate to detect the effect of the H215 amino acid residue on the catalytic activity of the BelK protein, and the specific steps are as follows:

1. synthesis of BelK H215A point mutant protein:

the synthesis process and method were the same as BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-H215A-F (5' -GAGTTTGCAGATGTG)GCAACCACCCTGTTTAGC-3 ') and BelK-H215A-R (5' -GCTAAACAGGGTGGT)TGCCACATCTGCAAACTC-3'), the results of electrophoresis are shown in FIG. 1.

2. Living test line of BelK H215A

TABLE 9 reaction system (Total volume 100. mu.L)

H was added in the order of Table 9₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK H215A protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2H.

3. Detection of the synthesized product

Adding 100 μ L acetonitrile into the reaction system to terminate the reaction, shaking and mixing, centrifuging at 13000rpm for 15min, collecting supernatant, adding 4 μ L dansyl chloride (DNSC, 50mM acetonitrile solution) to final concentration of 1mM, derivatizing in 50 deg.C constant temperature water bath for 1h, centrifuging at 13000rpm for 15min, adding sample bottle for HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK H215A assay in vivo lines, molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (fig. 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of H215 to alanine, and that the H215 amino acid residue had a significant effect on the catalytic function of BelK.

Example 9, this example uses the BelK E269A point mutant protein as catalytic enzyme, and uses L-lysine as substrate, and detects the effect of E269 amino acid residue on the catalytic activity of BelK protein, the specific steps are as follows:

1. synthesis of BelK E269A point mutant protein:

the procedure and method were the same as those of BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-E269A-F (5' -TATTACGGCGTGACC)GCATTTCTGGCACCGCGC-3 ') and BelK-E269A-R (5' -GCGCGGTGCCAGAAA)TGCGGTCACGCCGTAATA-3'), the results of electrophoresis are shown in FIG. 1.

2. Living assay line of BelK E269A

TABLE 10 reaction system (Total volume 100. mu.L)

H was added in the order of Table 10₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK E269A protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 hr.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK E269A in vivo assay, molecular weight [ M + H]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (figure 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of E269 to alanine, with the E269 amino acid residue having a significant effect on the catalytic function of BelK.

Example 10, in this example, the BelK H299A point mutant protein was used as a catalytic enzyme, L-lysine was used as a substrate, and the influence of the H299 amino acid residue on the catalytic activity of the BelK protein was examined, specifically, the following steps were performed:

1. synthesis of point mutant protein of BelK H299A:

the synthesis process and method were the same as BelK E205A in example 7, and the primer in BelK E205A was replaced with BelK-H299A-F (5' -GTGTACCACGACATC)GCAATCAACGTGGATGCC-3 ') and BelK-H299A-R (5' -GGCATCCACGTTGAT)TGCGATGTCGTGGTACAC-3'), the results of electrophoresis are shown in FIG. 1.

2. Living body testing line of BelK H299A

TABLE 11 reaction system (Total volume 100. mu.L)

H was added in the order of Table 11₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK H299A protein, and NADH are added finally, the mixture is mixed evenly to start reaction, and the mixture is placed for 2 hours at the temperature of 28 ℃.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK H299A assay in vivo line, a molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (figure 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of H299 to alanine, and that the H299 amino acid residues had a significant effect on the catalytic function of BelK.

Example 11, this example uses the BelK D303A point mutant protein as catalytic enzyme, uses L-lysine as substrate, detects the influence of D303 amino acid residue on the catalytic activity of BelK protein, and the concrete steps are as follows:

1. synthesis of BelK D303A point mutant protein:

the synthesis process and method were the same as BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-D303A-F (5' -ATCCACATCAACGTG)GCAGCCCACCATGCCGCC-3 ') and BelK-D303A-R (5' -GGCGGCATGGTGGGC)TGCCACGTTGATGTGGAT-3'), the results of electrophoresis are shown in FIG. 1.

2. Living organism testing line of BelK D303A

TABLE 12 reaction system (Total volume 100. mu.L)

H was added in the order of Table 12₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK D303A protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2 h.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK D303A assay in vivo lines, molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (fig. 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of D303 to alanine, and that the D303 amino acid residue had a significant effect on the catalytic function of BelK.

Example 12, this example uses the BelK H306A point mutant protein as a catalytic enzyme, and uses L-lysine as a substrate to detect the effect of the H306 amino acid residue on the catalytic activity of the BelK protein, and the specific steps are as follows:

1. synthesis of BelK H306A point mutant protein:

the synthesis process and method were the same as BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-H306A-F (5' -AACGTGGATGCCCAC)GCAGCCGCCGGTTGGTAT-3 ') and BelK-H306A-R (5' -ATACCAACCGGCGGC)TGCGTGGGCATCCACGTT-3'), the results of electrophoresis are shown in FIG. 1.

2. Living test line of BelK H306A

TABLE 13 reaction system (Total volume 100. mu.L)

H was added in the order of Table 13₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methyl sulfate, BelK H306A protein, and NADH, mixing, starting reaction, and standing at 28 deg.C for 2H.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK H306A assay in vivo lines, molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The product peak at 410 was still present (fig. 6), thus demonstrating that the catalytic function of BelK was completely lost after mutation of H306 to alanine, and that the H306 amino acid residue had a significant effect on the catalytic function of BelK.

Example 13, this example uses the BelK Y295A point mutant protein as catalytic enzyme, and uses L-lysine as substrate, and detects the effect of Y295 amino acid residue on the catalytic activity of BelK protein, the specific steps are as follows:

1. synthesis of BelK Y295A point mutant protein:

the synthesis process and method were the same as BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-Y295A-F (5' -CCTGAAGGTAAGGTG)GCACACGACATCCACATC-3 ') and BelK-Y295A-R (5' -GATGTGGATGTCGTG)TGCCACCTTACCTTCAGG-3'). The BelK Y295A point mutant protein is not soluble in the process of expression and purification, only exists in protein precipitation, and soluble protein is not detected in supernatant and purified solution, so that subsequent in vitro detection and product detection cannot be carried out. Thus, Y295 plays an important role in maintaining the normal tertiary structure of the BelK protein, and the mutation of the residue into alanine results in the failure of the protein to fold to form the correct higher-order structure, thereby causing the protein to precipitate.

Example 14, this example uses the BelK Y295F point mutant protein as catalytic enzyme, uses L-lysine as substrate, and detects the effect of Y295 amino acid residue on the catalytic activity of BelK protein, the specific steps are as follows:

1. synthesis of BelK Y295F point mutant protein:

the synthesis process and method were the same as BelK E205A in example 7, except that the primer in BelK E205A was replaced with BelK-Y295F-F (5' -CCTGAAGGTAAGGTG)TTTCACGACATCCACATC-3 ') and BelK-Y295F-R (5' -GATGTGGATGTCGTG)AAACACCTTACCTTCAGG-3'), the Y295F protein can form a correct fold compared to the Y295A point mutation, resulting in a soluble protein. The electrophoresis results are shown in FIG. 1.

2. Living test line of BelK Y295F

TABLE 14 reaction system (Total volume 100. mu.L)

H was added in the order of Table 14₂O、K₂HPO₄Buffer solution, L-lysine, phenazine methosulfate, BelK Y295F protein, adding NADH, mixingThe reaction was started and left at 28 ℃ for 2 h.

3. Detection of the synthesized product

Adding 100 mu L acetonitrile into a reaction system to terminate the reaction, shaking and mixing uniformly, centrifuging at 13000rpm for 15min, taking supernatant, adding 4 mu L dansyl chloride (dansyl chloride, DNSC, 50mM acetonitrile solution) to the final concentration of 1mM, placing in a constant temperature water bath kettle at 50 ℃ for derivatization for 1h, centrifuging at 13000rpm for 15min, adding a sample bottle, and carrying out HPLC-ESI- (HR) MS detection. Mobile phase: h₂O(0.1％HCOOH)+CH₃CN (0.1% HCOOH), flow rate of 1mL/min, ultraviolet absorption detection wavelength of 254nm, method: t is 0min, 5% B; t ═ 3min, 5% B; t18 min, 95% B; t22 min, 95% B; t23 min, 5% B; and T25 min, 5% B.

HPLC-ESI-MS showed a retention time of about 12.4min in the BelK Y295F assay in vivo lines, molecular weight [ M + H ]]⁺The product peak at 410 disappeared completely, while the positive control BelK assay in vivo retained for about 12.4min, molecular weight [ M + H]⁺The peak of the product at 410 still exists (fig. 6), which shows that the hydroxyl group of the benzene ring in Y295 can form a hydrogen bond with other amino acid residues to exert a normal catalytic function, and after Y295 is mutated to phenylalanine, the catalytic function of BelK is completely lost due to the deletion of the hydroxyl group.