Arsl mutant and application thereof in synthesis of arsinothricin

Abstract

This invention belongs to the field of bioengineering technology and relates to an ArsL mutant and its application in the synthesis of Arsinothricin. The ArsL mutant is a mutant obtained by mutating cysteine ​​C at amino acid position 428 of the wild-type AST synthase ArsL to alanine A. The amino acid sequence of the ArsL mutant is shown in SEQ ID No. 2. This invention further provides the application of the ArsL mutant in the catalytic synthesis of Arsinothricin from sodium methylarsenate and S-adenosylmethionine. The ArsL mutant of this invention exhibits higher catalytic activity and thermal stability. The high-concentration MMA to AST conversion process was achieved using the ArsL mutant. C428A The mutant exhibits significantly enhanced catalytic activity compared to the wild-type AST synthase. Furthermore, the yield using the synthetic pathway of this invention is significantly higher than that of organic synthetic pathways, semi-organic enzyme synthesis, and natural biosynthetic pathways. The ArsL mutant has broad application prospects as a potential enzyme for industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to an ArsL mutant and its application in the synthesis of Arsinothricin. Specifically, this application relates to an ArsL mutant and its encoded nucleic acid, a recombinant expression vector and recombinant expression transformant containing the ArsL mutant nucleic acid, a catalyst containing the ArsL mutant, and its application in the synthesis of Arsinothricin. Background Technology

[0002] Arsenic compounds have a long history of use as medicine. For example, arsenic trioxide remains the drug of choice for treating acute promyelocytic leukemia. Other organoarsenic compounds, including melarsoprol, atoxyl, and roxarsone, are used to treat cancer and microbial infections. Beyond humans, microorganisms in nature also utilize arsenic to synthesize antibiotics for protection. Arsenic-containing antibiotics can serve as novel antimicrobial agents to address the growing problem of antibiotic resistance in pathogenic bacteria.

[0003] Arsinothricin (AST) is a recently discovered broad-spectrum arsenic-containing antibiotic found in the culture medium of the soil bacterium Burkholderia gladioli in rice rhizospheres. It exhibits good inhibitory effects against both Gram-negative and Gram-positive bacteria, and also shows inhibitory activity against Plasmodium falciparum, the malaria parasite. Currently, the main methods for synthesizing AST are total organic synthesis and semi-organic enzymatic synthesis. These methods involve multiple chemical steps, are complex, have numerous procedures, and relatively low reaction efficiency.

[0004] The natural biosynthetic pathway of AST uses arsenic trioxide (arsenic trioxide) as a substrate. First, the ACP radical generated by the cleavage of S-adenosylmethionine (SAM) by ArsL adds to the arsenic atom, forming the intermediate AST-OH. Then, the SAM-dependent methyltransferase ArsM synthesizes AST-OH into AST.

[0005] Burkholderia gladioli ( Burkholderia gladioli ArsL, derived from AST, has been expressed and purified, and its catalytic activity for trihydroxyarsenic and its ability to cleave SAM to generate ACP radicals have been demonstrated. However, in the process of reproducing the natural biosynthetic pathway of AST in vitro, ArsL exhibits rather low catalytic activity for trihydroxyarsenic, and coupled with the requirement of two enzymatic reactions to complete the synthesis, its final synthetic efficiency is not ideal. Summary of the Invention

[0006] Both the organic total synthesis method and the semi-organic synthesis enzyme method for synthesizing the existing AST need chemical methods, and the process is complex, and the catalytic activity of ArsL to trihydroxy arsenic is low, and the synthesis efficiency is not high, based on the problem, the application provides an ArsL mutant and application thereof in synthesis of Arsinothricin. Specifically, the application relates to an ArsL mutant and a coding nucleic acid thereof, a recombinant expression vector and a recombinant expression transformant containing the nucleic acid of the ArsL mutant, a catalyst containing the ArsL mutant, and application thereof in synthesis of Arsinothricin.

[0007] The application realizes the application of the ArsL mutant in synthesis of Arsinothricin by protein engineering technology to molecularly modify the ArsL, and obtain the ArsL mutant with significantly improved activity.

[0008] The technical scheme of the application provides a new path for synthesizing Arsinothricin (AST), and the ArsL mutant can directly convert sodium methylarsenate (MMA) into AST, and the catalytic capacity of the mutant is significantly improved compared with the original ArsL, so that the mutant has a very wide application space for production of AST.

[0009] The purpose of the application can be achieved by the following technical scheme:

[0010] One of the technical schemes of the application is to provide an ArsL mutant, wherein the ArsL mutant is a mutant obtained by mutating cysteine C at the 428th amino acid in the amino acid sequence of a wild-type AST synthesis enzyme ArsL into alanine A, and the mutant is named as ArsL C428A .

[0011] The amino acid sequence of the wild-type AST synthesis enzyme ArsL is shown in SEQ ID No. 1; the wild-type AST synthesis enzyme ArsL is an S-adenosyl methionine radical enzyme, and is derived from Burkholderia glumae (B. glumae) (Burkholderia glumae), NCBI Reference Sequence: WP_219608243.1. Burkholderia gladioli

[0012] The nucleotide sequence of the coding gene of the wild-type AST synthesis enzyme ArsL is shown in SEQ ID No. 3.

[0013] In one embodiment of the application, the amino acid sequence of the ArsL mutant is shown in SEQ ID No. 2.

[0014] The ArsL mutant provided by the application C428A can directly convert sodium methylarsenate (MMA) into AST, and the catalytic capacity of the mutant is significantly improved compared with the wild-type AST synthesis enzyme ArsL. ​

[0015] Further, the method for obtaining the mutant is specifically as follows:

[0016] The gene sequence of wild-type AST synthetase ArsL in Burkholderia glumae is used as a template, and a mutant library is constructed by using site-directed saturation mutation. Burkholderia gladioli The mutant library is screened by high-throughput screening, and the mutant is obtained. The high-throughput screening refers to random mutation or single-point saturation mutation of the wild-type AST synthetase ArsL, activity detection by using a substrate sodium methylarsenate (MMA), and screening of mutants with significantly improved activity.

[0017] The whole plasmid PCR amplification of the site-directed saturation mutation library is a conventional technology in the field, and the system and program of the PCR amplification reaction can be obtained by using conventional technical means in the field.

[0018] The second technical solution of the present application is to provide an isolated nucleic acid encoding the ArsL mutant in the first technical solution.

[0019] Further, the preparation method of the nucleic acid is a conventional preparation method in the field, and the preparation method comprises: obtaining a nucleic acid molecule encoding the ArsL mutant by gene cloning technology, or obtaining a nucleic acid molecule encoding the ArsL mutant by an artificial full-sequence synthesis method; preferably, the DNA sequence of the ArsL mutant obtained in the first technical solution is used as a template, and the nucleic acid molecule with a point mutation is obtained by PCR amplification of the target gene.

[0020] The PCR amplification technology is a conventional technology in the field, and the system and program of the PCR amplification reaction can be obtained by using conventional technical means in the field.

[0021] In an embodiment of the present application, the nucleotide sequence of the isolated nucleic acid (ArsL C428A The nucleotide sequence of the synthetic gene of the ArsL mutant is shown in SEQ ID No. 4.

[0022] The third technical solution of the present application is to provide a recombinant expression vector comprising the above nucleic acid.

[0023] Further, the recombinant expression vector can be obtained by a conventional method in the field, that is, the nucleic acid molecule of the synthetic gene of the ArsL mutant is connected to various commercially available expression vectors to construct the recombinant expression vector; the expression vector is preferably a plasmid pET-28a(+). For example, the recombinant expression plasmid in the present application can be prepared by the following method: the nucleic acid product obtained by PCR amplification and the expression vector pET-28a(+) are respectively digested by restriction enzymes NdeI andEcoRI Double digestion with enzymes forms complementary sticky ends, which are then ligated using a ligase to form a recombinant expression plasmid containing the ArsL mutant gene described in this invention. Specifically, the recombinant vector is formed by replacing the DNA fragments in the NdeI and EcoRI recognition sequences of the pET28a(+) vector with ArsL. C428A Recombinant plasmids obtained from expressing genes.

[0024] Using the same method, the DNA fragments in the NdeI and EcoRI recognition sequences of the pET28a(+) vector can be replaced with the ArsL expression gene to obtain a recombinant plasmid. The ArsL expression gene sequence is shown in SEQ ID NO.3. C428A The expressed gene sequence is shown in SEQ ID NO.4, and the ArsL C428A The expression gene was obtained by mutating TG to GC at positions 1282-1283 of the ArsL expression gene.

[0025] The fourth technical solution adopted by the present invention is to provide a recombinant expression transformant containing the above-mentioned recombinant expression vector.

[0026] The preferred method for preparing the recombinant expression transformant is to transform the above-mentioned recombinant expression vector into a host microorganism; the host microorganism is preferably *Escherichia coli*. Escherichia coli abbreviated as E. coli More preferably, it is *Escherichia coli* BL21 (DE3). The aforementioned recombinant expression vector is transformed into *Escherichia coli*. E. coli In BL21 (DE3), the preferred recombinant expression transformant of the present invention (a genetically engineered strain) is obtained. The plasmid transformation method can be any conventional method in the art, such as electroporation, heat shock, etc.

[0027] The fifth technical solution adopted by the present invention is to provide an ArsL mutant catalyst, wherein the catalyst can be any of the following forms:

[0028] (1) Cultivate the recombinant expression transformant of the present invention and isolate the transformant cells containing the ArsL mutant, i.e., the resting cells of the recombinant expression transformant;

[0029] (2) Cultivate the recombinant expression transformant of the present invention, isolate the transformant cells containing the ArsL mutant and break them to obtain the lysate, or obtain the pure enzyme solution after the lysate is purified and separated.

[0030] (3) Cultivate the recombinant expression transformant of the present invention, isolate the transformant cells containing the ArsL mutant and break them to obtain a lysate, and freeze-dry the lysate to obtain enzyme powder.

[0031] The preparation method of the mutant ArsL catalyst is preferably:

[0032] The ArsL C428A The single colony of the expression bacteria is picked and cultured in 10 mL of LB medium containing 50 micrograms / mL of kanamycin overnight. Then it is transferred into 1 liter of LB medium containing 50 micrograms / mL of kanamycin for expansion culture until the OD 600 of the bacterial solution reaches 0.6-0.8. 200 microliters of 1M IPTG is added to induce the expression of the expression gene at 18°C and 100 rpm. The required expression time is 18 hours.

[0033] After the expression is completed, the bacterial solution is centrifuged to collect, the supernatant culture medium is completely removed, and the bacteria are resuspended with 30 mL of 50 mM Tris-HCl (pH 8.0) buffer. In an anaerobic glove box, the bacteria are broken using an ultrasonic disrupter, and the cell debris is removed by centrifugation, and the supernatant is the lysate of the recombinant cells expressing ArsL C428A . The lysate is freeze-dried to obtain enzyme powder.

[0034] The purification operation is an operation that can be easily understood and implemented by those skilled in the art, and is preferably a method of Ni column affinity chromatography; the freeze-drying refers to pre-cooling the crude enzyme solution and placing it in a freeze dryer for freeze-drying for about 30-80 h to obtain freeze-dried enzyme powder.

[0035] Based on this, the application further provides a catalyst based on the wild-type AST synthetase ArsL, which can be in any of the following forms:

[0036] (1) culturing the recombinant expression transformant of the wild-type AST synthetase ArsL, and isolating the transformant cells containing the wild-type AST synthetase ArsL, i.e., the resting cells of the recombinant expression transformant;

[0037] (2) culturing the recombinant expression transformant of the wild-type AST synthetase ArsL, and isolating the lysate obtained after the transformant cells containing the wild-type AST synthetase ArsL are broken, or the pure enzyme solution obtained after the lysate is subjected to a purification and isolation operation;

[0038] (3) culturing the recombinant expression transformant of the wild-type AST synthetase ArsL, isolating the lysate obtained after the transformant cells containing the wild-type AST synthetase ArsL are broken, and freeze-drying the enzyme powder obtained from the lysate.

[0039] The sixth technical solution of the present application is to provide the application of wild-type AST synthetase ArsL, the mutant of the ArsL, the catalyst based on the wild-type AST synthetase ArsL or the mutant catalyst of the ArsL in catalyzing the synthesis of Arsinothricin from sodium methylarsenate (MMA) and S-adenosyl methionine (SAM).

[0040] In one embodiment of the present application, the wild-type AST synthetase ArsL, the mutant of the ArsL, the catalyst based on the wild-type AST synthetase ArsL or the mutant catalyst of the ArsL are used to catalyze the reaction with sodium methylarsenate (MMA) and S-adenosyl methionine (SAM) as substrates, dithiothreitol (DTT) as an arsenic reducing agent, and titanium (III) citrate as a reducing agent of the iron-sulfur cluster in the ArsL enzyme, to obtain Arsinothricin (AST).

[0041] In one embodiment of the present application, hydrogen peroxide solution and trifluoroacetic acid are added to terminate the reaction after the reaction is completed.

[0042] In one embodiment of the present application, the reaction system for catalyzing the reaction can be obtained by adding SAM, DTT, MMA and titanium (III) citrate to 200 microliters of enzyme solution of wild-type AST synthetase ArsL or the mutant of the ArsL, and then adding 50 mM Tris hydrochloric acid buffer (pH 8.0) to 300 microliters. The final concentrations of SAM, DTT, MMA and titanium (III) citrate in the reaction system are 10-20 mM, 10-20 mM, 5-15 mM and 10-20 mM, respectively.

[0043] In one embodiment of the present application, the reaction system for catalyzing the reaction can be obtained by adding SAM, DTT, MMA and titanium (III) citrate to 200 microliters of enzyme solution of wild-type AST synthetase ArsL or the mutant of the ArsL, and then adding 50 mM Tris hydrochloric acid buffer (pH 8.0) to 300 microliters. The final concentrations of SAM, DTT, MMA and titanium (III) citrate in the reaction system are 12 mM, 15 mM, 10 mM and 15 mM, respectively. After the reaction is completed, 10 microliters of 30% hydrogen peroxide solution and 10 microliters of trifluoroacetic acid are added to terminate the reaction.

[0044] The seventh technical solution of the present application: a method for synthesizing Arsinothricin is provided, taking MMA and SAM as substrates, DTT as an arsenic reducing agent, titanium (III) citrate as a reducing agent for iron-sulfur clusters in ArsL enzyme, and using wild-type AST synthetase ArsL, the ArsL mutant, the catalyst based on wild-type AST synthetase ArsL or the ArsL mutant catalyst to catalyze the reaction, so as to obtain Arsinothricin.

[0045] Compared with the prior art, the present application has the following advantages and effects:

[0046] (1) The ArsL mutant has higher catalytic activity and thermal stability, and the process of converting high-concentration MMA to AST is realized by using the ArsL mutant, and the ArsL mutant has higher catalytic activity than the wild-type AST synthetase. C428A The catalytic activity of the mutant is significantly higher than that of the wild-type AST synthetase.

[0047] (2) The organic total synthesis method and the semi-organic synthesis enzyme method used in the prior art for synthesizing AST both need chemical methods, and the process is complex, while the enzyme catalyst catalytic reaction provided by the present application has the advantages of mild reaction conditions, safe operation, green and environmentally friendly process, etc., and has good industrial application prospect. And the yield of this synthesis path is significantly higher than that of the organic synthesis path, semi-organic enzyme synthesis and natural biosynthesis path, and the ArsL mutant can be used as a potential industrial application enzyme, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a path diagram for synthesizing AST designed in the present application.

[0049] Figure 2 It is a test diagram of the bacteriostatic effect of AST. The left graph is Escherichia coli, the left point is a blank control, and the right point is an inhibition ring produced by AST on the LB solid medium for the growth of Escherichia coli. The right graph is Staphylococcus aureus, the left point is a blank control, and the right point is an inhibition ring produced by AST on the LB solid medium for the growth of Staphylococcus aureus.

[0050] Figure 3 It is an HR-MSMS spectrum of AST.

[0051] Figure 4 It is a mass spectrum signal comparison diagram of AST in the reaction products of different catalytic systems in the present application.

[0052] Figure 5 It is a mass spectrum signal comparison diagram of MMA(V) in the reaction products of different catalytic systems in the present application. DETAILED DESCRIPTION

[0053] For those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in combination with specific examples. It should be pointed out that the following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0054] Unless otherwise specified, the specific experiments in the following examples are carried out according to the conventional methods and conditions in the art, or according to the commercial instructions of the reagent kits.

[0055] Sodium methylarsenate: Shanghai Bailingwei Chemical Technology Co., Ltd., item number P-279N;

[0056] The plasmid pET28a-ArsL is constructed by the inventors according to the conventional methods in the art;

[0057] The present application will be described in detail below in combination with the drawings and specific examples.

[0058] Example 1

[0059] ArsL C428A Construction of expression plasmid

[0060] The plasmid pET28a-ArsL is used as a template, and the primers in Table 1 are used as upstream and downstream primers respectively to construct by PCR. The PCR program is as follows: pre-denaturation at 98℃ for 3 minutes, denaturation at 98℃ for 15s, annealing at 55℃ for 15s, extension at 72℃ for 7 minutes, and finally at 72℃ for 10 minutes. The denaturation to extension program is set to 25 cycles.

[0061] Table 1 Primers for modifying ArsL

[0062]

[0063] The sequence of ArsL C428A F is shown in SEQ ID NO. 5, and the sequence of ArsL C428A R is shown in SEQ ID NO. 6.

[0064] Dpn I enzyme was added to the PCR product for 1 microliter to eliminate the original plasmid template, and the digestion was performed overnight in a 37°C constant temperature water bath to completely digest the original template. 5 microliters of the enzyme-digested PCR product was added to 100 microliters of E. coli DH5α competent cells, incubated on ice for 30 minutes, then heated in a 42°C water bath for 1 minute, and then transferred to ice for 3 minutes. 800 microliters of LB medium was added, and the culture was incubated in a 37°C constant temperature incubator for 1 hour. The cells were collected by centrifugation, most of the supernatant was discarded, the cells were resuspended with the remaining medium, and the bacterial liquid was spread on a kanamycin-resistant (concentration of 50 micrograms / milliliter) LB plate, and incubated in a 37°C constant temperature incubator until single colonies were grown. The single colonies on the plate were picked out for sequencing to verify that the mutation was complete. Then the plasmid extraction kit was used to extract the ArsL C428A expression plasmid.

[0065] 5 microliters of the ArsL C428A expression plasmid was added to 100 microliters of E. coli BL21 (DE3) competent cells, incubated on ice for 30 minutes, then heated in a 42°C water bath for 1 minute, and then transferred to ice for 3 minutes. 800 microliters of LB medium was added, and the culture was incubated in a 37°C constant temperature incubator for 1 hour. The cells were collected by centrifugation, most of the supernatant was discarded, the cells were resuspended with the remaining medium, and the bacterial liquid was spread on a kanamycin-resistant (concentration of 50 micrograms / milliliter) LB plate, and incubated in a 37°C constant temperature incubator until single colonies were grown. The single colonies on the plate were picked out for sequencing to verify that the mutation was complete. Then the plasmid extraction kit was used to extract the ArsL C428A expression bacteria.

[0066] In the same way, the original ArsL expression plasmid was transformed into E. coli BL21 (DE3) competent cells to obtain ArsL expression bacteria.

[0067] Example 2

[0068] ArsL and ArsL C428A expression

[0069] First, single colonies of ArsL or ArsL C428A expression bacteria were picked out and cultured overnight in 10 mL of LB medium containing 50 micrograms / milliliter of kanamycin. Then they were transferred to 1 liter of LB medium containing 50 micrograms / milliliter of kanamycin for expansion culture until the OD 600 of the bacterial liquid reached 0.6-0.8. 200 microliters of 1M IPTG was added, and the expression of the expression gene was induced at 18°C and 100 rpm. The required expression time was 18 hours.

[0070] After expression was completed, the bacterial solution was centrifuged to collect, completely remove the supernatant culture medium, and resuspend the bacteria with 30 mL of 50 mM Tris-HCl (pH 8.0) buffer. In the glove box, the bacteria were broken using an ultrasonic disruptor, and the cell debris was removed by centrifugation. The supernatant was the lysate of the recombinant cells expressing ArsL or ArsL C428A .

[0071] Example 3

[0072] Purification of ArsL and ArsL C428A

[0073] The lysate obtained in Example 2 was transferred to a Ni-NTA resin column, so that the target enzyme was adsorbed on the nickel column. The impurities were washed away using 50 mM Tris-HCl (pH 8.0) buffer, and the target protein was eluted using 50 mM Tris-HCl (pH 8.0) buffer containing 500 mM imidazole.

[0074] In the process of reconstituting the iron-sulfur cluster, first add DTT to 10 mM, then add ferrous sulfate to 1 mM in two times, with an interval of 20 minutes each time. Add sodium sulfide to 1 mM in two times, with an interval of 20 minutes each time. After incubation on ice overnight, remove the precipitate with a centrifuge, and desalt the supernatant on a PD-10 column (Bio-Rad). The PD-10 column is pre-equilibrated with desalting buffer (50 mM Tris, 25 mM NaCl and 10% glycerol, pH 8.0), and the target monomer is also eluted with the desalting buffer.

[0075] Example 4

[0076] Bacteriostatic effect of arsenothricin (AST) as a broad-spectrum antibiotic

[0077] The test diagram of the bacteriostatic effect of AST is shown in Figure 2 , where the left graph is Escherichia coli, the left dot is a blank control, and the right dot is an inhibition ring produced by AST on the LB solid medium in which Escherichia coli grows. The right graph is Staphylococcus aureus, the left dot is a blank control, and the right dot is an inhibition ring produced by AST on the LB solid medium in which Staphylococcus aureus grows. It can be seen that AST has good inhibition effect on Escherichia coli and Staphylococcus aureus.

[0078] The HR-MSMS spectrum of AST is shown in Figure 3 .

[0079] Example 5

[0080] Synthesis of AST

[0081] Reference Figure 1 ​, MMA and SAM as substrates, DTT as arsenic reductant, and titanium citrate (III) as reductant of iron-sulfur cluster in ArsL enzyme, using wild-type AST synthetase ArsL or ArsL mutant ArsL C428A Arsinothricin was obtained by catalytic reaction.

[0082] ArsL or ArsL mutant was added into 200 μL of enzyme solution, and then 50 mM Tris-HCl buffer (pH 8.0) was added to make the volume to 300 μL. The final concentrations of SAM, DTT, MMA and titanium citrate (III) in the reaction system were 12 mM, 15 mM, 10 mM and 15 mM, respectively. C428A ArsL or ArsL mutant was added into 200 μL of enzyme solution, and then 50 mM Tris-HCl buffer (pH 8.0) was added to make the volume to 300 μL. The final concentrations of SAM, DTT, MMA and titanium citrate (III) in the reaction system were 12 mM, 15 mM, 10 mM and 15 mM, respectively.

[0083] The reaction was carried out in an anaerobic glove box, and the reaction time was 2 hours. After the reaction was completed, 10 μL of 30% hydrogen peroxide solution and 10 μL of trifluoroacetic acid were added to terminate the reaction, and the product was released from the enzyme. The amounts of AST and MMA were detected in the product by mass spectrometry.

[0084] ArsL or ArsL mutant was added into 200 μL of enzyme solution, and then 50 mM Tris-HCl buffer (pH 8.0) was added to make the volume to 300 μL. The final concentrations of SAM, DTT, MMA and titanium citrate (III) in the reaction system were 12 mM, 15 mM, 10 mM and 15 mM, respectively.

[0085] The reaction was carried out in an anaerobic glove box, and the reaction time was 2 hours. After the reaction was completed, 10 μL of 30% hydrogen peroxide solution and 10 μL of trifluoroacetic acid were added to terminate the reaction, and the product was released from the enzyme. The amounts of AST and MMA were detected in the product by mass spectrometry. Figure 4 Figure 5 The amounts of AST and MMA were detected in the product by mass spectrometry. For AST, the signal intensity at the mass-to-charge ratio of 226.00-226.01 was detected; for MMA, the signal intensity at the mass-to-charge ratio of 140.95-140.96 was detected. The specific signal spectrum is shown in FIGS. 1 and 2.

[0086] The signal of AST in the catalytic product is shown in Table 2, and the signal of MMA in the catalytic product is shown in Table 3.

[0087] Table 2 Signal of AST in the catalytic product

[0088]

[0089] Table 3 Signal of MMA in the catalytic product ​

[0090]

[0091] The results are shown in Tables 2 and 3. ArsL C428A The catalytic ability for MMA was significantly improved, and 10 mM of MMA was completely converted into AST. After the reaction was completed, the signal of the raw material substrate MMA could not be detected in the mass spectrum. The catalytic activity of the lysate was relatively low compared to the enzyme solution, but the purification step was omitted, which was conducive to large-scale reactions.

[0092] In summary, the mutant ArsL was used to realize the conversion of high-concentration MMA to AST. ArsL C428A The catalytic activity of the mutant was significantly improved compared to the original ArsL. Moreover, the yield using this synthesis path was significantly improved compared to the catalytic yield of the organic synthesis path, semi-organic enzyme synthesis, and natural biosynthesis path, and it could be used as a potential industrial application enzyme.

[0093] The above description of the embodiments is to facilitate the understanding and use of the invention by those of ordinary skill in the art. Those skilled in the art can obviously make various modifications to these embodiments, and apply the general principles described herein to other embodiments without having to go through creative labor. Therefore, the present application is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present application without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. An ArsL mutant, characterized in that, The ArsL mutant is a mutant obtained by mutating cysteine ​​C at amino acid sequence 428 of the wild-type AST synthase ArsL to alanine A, and the amino acid sequence of the ArsL mutant is shown in SEQ ID No.

2.

2. An isolated nucleic acid, characterized in that, The nucleic acid encodes the ArsL mutant as described in claim 1, and the nucleotide sequence of the isolated nucleic acid is shown in SEQ ID No.

4.

3. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 2.

4. A recombinant expression transformant, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. An ArsL mutant catalyst, characterized in that, It is any of the following forms: (1) Cultivate the recombinant expression transformant according to claim 4, and isolate the transformant cells containing the ArsL mutant, i.e., the resting cells of the recombinant expression transformant; (2) Cultivate the recombinant expression transformant of claim 4, isolate the transformant cells containing the ArsL mutant and break them to obtain a lysate, or obtain a pure enzyme solution after the lysate has been purified and separated. (3) Cultivate the recombinant expression transformant according to claim 4, isolate the transformant cells containing the ArsL mutant and break them to obtain a lysate, and freeze-dry the lysate to obtain enzyme powder.

6. The use of the ArsL mutant of claim 1 or the ArsL mutant catalyst of claim 5 in the catalytic synthesis of Arsinothricin from sodium methylarsenate and S-adenosylmethionine.

7. The application as described in claim 6, characterized in that, Arsinothricin was obtained by using sodium methylarsenate and S-adenosylmethionine as substrates and the ArsL mutant or ArsL mutant catalyst for catalytic reaction, with dithiothreitol as arsenic reducing agent and titanium citrate as reducing agent for iron-sulfur clusters in ArsL enzyme.

8. The application as described in claim 7, characterized in that, After the reaction is completed, hydrogen peroxide solution and trifluoroacetic acid are added to terminate the reaction.

9. The application as described in claim 7, characterized in that, The final concentrations of S-adenosylmethionine, dithiothreitol, sodium methylarsenate, and titanium citrate in the reaction system were 10-20 mM, 10-20 mM, 5-15 mM, and 10-20 mM, respectively. Based on a reaction system volume of 300-400 μL, the amount of the ArsL mutant enzyme solution used in the reaction system is 200 μL.

Images