Method for purifying dihydroxy acid dehydratase and determining enzyme activity

Abstract

The invention discloses a purification and enzyme activity determination method of dihydroxy acid dehydratase, and belongs to the technical field of enzyme activity determination. According to the method for purifying the dihydroxy acid dehydratase and determining the enzyme activity, disclosed by the invention, the dihydroxy acid dehydratase obtained through in-vitro reconstruction of iron-sulfur clusters and purification can keep stable enzyme activity on the basis of a laboratory reduction method innovatively. Pyruvic acid is selected as a substrate, and generation of a catalytic precursor substance 2, 3-dihydroxy isovaleric acid of the dihydroxy acid dehydratase and synthesis of a catalytic product alpha-ketoisovaleric acid can be realized by finely designing a multi-enzyme cascade reaction; through high performance liquid chromatography analysis, an external standard method is adopted for quantification, the synthesis amount of alpha-ketoisovaleric acid is measured, and the enzyme activity is indirectly calculated. The determination method provided by the invention is simple to operate and low in operation environment requirement, and the operation cost is greatly reduced while the accuracy and the stability are considered.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme activity determination, and more particularly to a purification and enzyme activity determination method of dihydroxy-acid dehydratase. BACKGROUND

[0002] Dihydroxy-acid dehydratase (DHAD) is a key enzyme in the third step of branched-chain amino acid (leucine, isoleucine, valine) synthesis pathway, which can catalyze 2,3-dihydroxy-isovalerate (DHIV) to generate α-ketoisovalerate (2-ketoisovalerate, KIV). At present, there are few studies on this enzyme using computational simulation strategy, on the one hand because the experimental structure of the enzyme is limited, and on the other hand because the catalytic site of the enzyme is complex, which needs Fe-S cluster and Mg 2+ cooperation. It is known that the Fe-S cluster contained in DHAD includes two types, [4Fe-4S] and [2Fe-2S], taking DHAD from E. coli and DHAD from S. aureus as examples, respectively. The [4Fe-4S] cluster is easily degraded by oxygen, thereby affecting the activity of the enzyme.

[0003] Using the current commonly used purification method to purify [4Fe-4S] dependent dihydroxy-acid dehydratase, it is difficult to effectively maintain the catalytic activity of the enzyme, and the purification and catalytic process require a high-oxygen-free environment and complex technical operation. The existing dihydroxy-acid dehydratase enzyme activity determination is to use the precursor 2,3-dihydroxy-isovalerate to directly catalyze the synthesis of α-ketoisovalerate. In this method, the substrate 2,3-dihydroxy-isovalerate is expensive, and the cost of enzyme activity determination is extremely high, which limits the research on dihydroxy-acid dehydratase.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a purification and enzyme activity determination method of dihydroxy-acid dehydratase. SUMMARY

[0005] Therefore, the present application provides a purification and enzyme activity determination method of dihydroxy-acid dehydratase, which can maintain the stability of the catalytic properties of dihydroxy-acid dehydratase and realize low-cost enzyme activity determination through in vitro reconstitution of iron-sulfur cluster purification and three-step cascade reaction.

[0006] In order to achieve the above purpose, the present application adopts the following technical solutions: A purification and enzyme activity determination method of dihydroxy-acid dehydratase, comprising the following steps: (1) Purification of dihydroxy-acid dehydratase The crude enzyme solution of dihydroxy acid dehydratase is placed in ice water, 1.0-1.4 mg / mL of protamine sulfate and 0.5-2 mM of 1,4-dithiothreitol are added, and after stirring and mixing, the reaction is carried out on ice for 10-20 min, centrifugation is carried out at 12000 rpm for 10-15 min, and the supernatant is reserved; the supernatant is naturally flowed in a nickel column, so that the protein is adsorbed on the filler of the nickel column, and 50 mM of imidazole solution is used to elute the impurities, and the operation is repeated 1-3 times; 100 mM of imidazole solution is used to elute the target protein adsorbed in the nickel column, and the elution is carried out 2-3 times, and the filtrate is collected; 0.1-1 mM of 1,4-dithiothreitol is added to the filtrate again, and ultrafiltration is used for concentration; desalination is carried out on a Nap column, and the target pure enzyme is obtained.

[0007] (2) Enzyme activity determination of dihydroxy acid dehydratase 100 µg of acetolactate synthase is added to 10 mL of pyruvic acid reaction solution, and the reaction time is 10 min; after the reaction is completed, 100 µg of ketonic acid reduction isomerase and 1 mL of 0.5-1 mM of NAD(P)H solution are added, and the reaction time is 10 min, and the substrate solution is obtained; 100 µg of dihydroxy acid dehydratase is added to 9.2 mL of the substrate solution, 0.8 mL of the reconstituted iron-sulfur cluster solution is added, and the reaction is carried out for 10 min to synthesize α-ketoisovaleric acid; 1 mL of 2M hydrochloric acid solution is added to terminate the reaction, and the enzymatic reaction liquid test sample solution is obtained; 0.8 mL of the reconstituted iron-sulfur cluster solution is added to 9.2 mL of the substrate solution, and after mixing, it is left to stand for 10 min, and then 1 mL of 2M hydrochloric acid solution is added, and the blank control solution is obtained.

[0008] The pyruvic acid reaction solution is 10-20 mM of pyruvic acid, 0.5-2 mM of thiamine pyrophosphate, 10-20 mM of MgCl2, and the solvent is Tris-HCl buffer solution c.

[0009] The reconstituted iron-sulfur cluster solution is 10-20 mM of MgCl2, 10-20 mM of FeSO4, 10-20 mM of L-cysteine, and 0.5-2 mM of 1,4-dithiothreitol, and the solvent is ultrapure water.

[0010] The standard curve is established by high-performance liquid chromatography, with the concentration gradient of the α-ketoisovaleric acid standard as the abscissa and the peak area as the ordinate, and the test sample solution and the blank control solution are analyzed to calculate the enzyme activity.

[0011] The standard curve is y=ax+b=14312x-49.008.

[0012] The enzyme activity calculation formula is: Enzyme activity = [(A0- A1) x N x V] / [m x t x M x a].

[0013] Wherein a is the slope of the standard curve, b is the intercept of the standard curve; A0is the peak area of a-ketoisovaleric acid in the blank control solution; A1is the peak area of a-ketoisovaleric acid in the solution after the reaction; V is the total volume of the reaction system; m is the mass of dihydroxy acid dehydratase sample; t is the reaction time of the enzyme and the substrate; M is the molar mass of a-ketoisovaleric acid; N is the dilution multiple of the enzyme solution to the test solution.

[0014] Further, the dihydroxy acid dehydratase in step (1) is wild type and mutant of dihydroxy acid dehydratase; The mutant of dihydroxy acid dehydratase is A268D or C195M; The amino acid sequence of the wild type of dihydroxy acid dehydratase is shown in SEQ ID NO. 1.

[0015] Further, the preparation method of the crude enzyme solution of dihydroxy acid dehydratase in step (1) is as follows: The recombinant plasmid after connecting the gene fragment of dihydroxy acid dehydratase with His tag to the vector pET28a is transformed into E. coli BL21, and the correct transformant is cultured to OD 600 When the OD is 0.6-0.8, 0.05-0.2 mM IPTG is added to induce expression at 16°C for 12-18 h, then the cells are resuspended with 15-20 mL of Tris-HCl buffer a, the cells are broken, the supernatant is obtained after centrifugation, and the crude enzyme solution is obtained; The nucleotide sequence with His tag is shown in SEQ ID NO. 2; and the gene fragment of dihydroxy acid dehydratase is shown in any one of SEQ ID NO. 3-5.

[0016] Further, the 50 mM imidazole solution is 0.34 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The 100 mM imidazole solution is 0.68 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer a is 50 mM Tris reagent, 10-30 mM MgCl2, the solvent is ultrapure water, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer b is 50 mM Tris reagent, 150 mM NaCl, 10-30 mM MgCl2, the solvent is ultrapure water, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer c is 50 mM Tris reagent, the solvent is ultrapure water, and the pH is adjusted to 8.0 by using 12M hydrochloric acid.

[0017] Further, the high performance liquid chromatography condition in step (2) is that the mobile phase is 0.5 mM sulfuric acid, the flow rate of the mobile phase is 0.3-0.8 mL / min, and the detection wavelength is 315-340 nm.

[0018] Compared with the prior art, the application provides a dihydroxy acid dehydratase purification and enzyme activity determination method and a dihydroxy acid dehydratase purification method, which has low operation difficulty and low technical requirement and has more stable enzyme catalytic properties. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0020] Figure 1 The pET28a expression plasmid pET28a-ilvD carrying the His-tagged dihydroxy acid dehydratase encoding gene ilvD.

[0021] Figure 2 The pET28a expression plasmid pET28a-ilvD carrying the His-tagged dihydroxy acid dehydratase encoding gene ilvD A268D .

[0022] Figure 3 The pET28a expression plasmid pET28a-ilvD carrying the His-tagged dihydroxy acid dehydratase encoding gene ilvD C195M .

[0023] Figure 4 The three-step cascade catalytic reaction of dihydroxy acid dehydratase; A is the first step reaction: catalytic synthesis of 2-acetyllactic acid from pyruvic acid; B is the second step reaction: catalytic synthesis of 2,3-dihydroxyisovaleric acid from 2-acetyllactic acid; C is the third step reaction: catalytic synthesis of alpha-ketoisovaleric acid from 2,3-dihydroxyisovaleric acid.

[0024] Figure 5 The standard curve of alpha-ketoisovaleric acid. DETAILED DESCRIPTION

[0025] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0026] The amino acid sequence of the wild-type dihydroxy acid dehydratase is shown in SEQ ID NO. 1.

[0027] MPKYRSATTTHGRNMAGARALWRATGMTDADFGKPIIAVVNSFTQFVPGHVHLRDLGKLVAEQIEAAGGVAKEFNTIAVDDGIAMGHGGMLYSLPSRELIADSVEYMVNAHCADAMVCISNCDKITPGMLMASLRLNIPVIFVSGGPMEAGKTKLSDRIIKLDLVDAMIQGADPKVSDSQSDQVERSACPTCGS C SGMFTANSMNCLTEALGLSQPGNGSLLATHADRKQLFLNAGKRIVELTKRYYEQNDESALPRNIASKAAFEN A MTLDIAMGGSTNTVLHLLAAAQEAEIDFTMSDIDKLSRKVPQLCKVAPSTQKYHMEDVHRAGGVIGILGELDRAGLLNRDVKNVLGLTLPQTLEQYDVMLTQDDAVKNMFRAGPAGIRTTQAFSQDCRWDSLDDDRANGCIRSLEHAYSKDGGLAVLYGNFAENGCIVKTAGVDDSILKFTGPAKVYESQDDAVEAILGGKVVAGDVVVIRYEGPKGGPGMQEMLYPTSFLKSMGLGKACALITDGRFSGGTSGLSIGHVSPEAASGGSIGLIEDGDLIAIDIPNRGIQLQVSDAELAARREAQEARGDKAWTPKNRERQVSFALRAYASLATSADKGAVRDKSKLGG; SEQ ID NO. 1.

[0028] The nucleotide sequence with His tag is shown in SEQ ID NO. 2.

[0029] CGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGA CTGGTGGACAGCAAATGGGTCGC ; SEQ ID NO. 2.

[0030] The nucleotide sequence of the wild-type dihydroxy acid dehydratase-encoding gene ilvD is shown in SEQ ID NO. 3.

[0031] ATGCCTAAGTACCGTTCCGCC ACCACCACCCATGGCCGTAATATGGCGGGGGCCCGCGCACTGTGGCGCGCCACCGGGATGACCGACGCCGATTTCGGTAAGCCGATTATCGCGGTTGTGAACTCGTTCACCCAATTTGTACCGGGTCACGTCCATCTGCGCGATCTCGGTAAACTGGTCGCCGAACAAATTGAAGCGGCTGGCGGCGTTGCCAAAGAGTTCAACACCATTGCGGTGGATGATGGGATTGCCATGGGCCACGGGGGGATGCTTTATTCACTGCCATCTCGCGAACTGATCGCTGATTCCGTTGAGTATATGGTCAACGCCCACTGCGCCGATGCCATGGTCTGTATCTCCAACTGCGACAAAATCACCCCTGGGATGTTGATGGCTTCCCTGCGATTAAATATTCCGGTGATCTTTGTTTCCGGCGGCCCGATGGAAGCCGGGAAAACCAAGCTGTCCGATCGGATAATCAAGCTCGATCTGGTTGATGCGATGATCCAGGGCGCAGACCCGAAAGTCTCTGACTCCCAGAGCGATCAGGTTGAACGTTCCGCCTGCCCAACCTGCGGTTCC TGCTCCGGGATGTTTACCGCTAACTCAATGAACTGCCTGACCGAAGCGCTGGGTTTGTCGCAGCCAGGCAACGGCTCGCTGCTGGCAACCCACGCGGACCGTAAGCAGCTGTTCCTTAATGCTGGTAAACGCATTGTTGAATTGACCAAACGTTATTACGAGCAAAACGACGAAAGTGCACTGCCGCGTAATATCGCCAGTAAGGCGGCGTTTGAAAAC GCCGCGATAAATCGAAACTGGGGGGTTAA ; SEQ ID NO. 3.

[0032] Genes encoding dihydroxy acid dehydratase mutants with mutation site A268D A268D The nucleotide sequence is shown as SEQ ID NO. 4.

[0033] ATGCCTAAGTACCGTTCCGCC ACCACCACCCATGGCCGTAATATGGCGGGGGCCCGCGCACTGTGGCGCGCCACCGGGATGACCGACGCCGATTTCGGTAAGCCGATTATCGCGGTTGTGAACTCGTTCACCCAATTTGTACCGGGTCACGTCCATCTGCGCGATCTCGGTAAACTGGTCGCCGAACAAATTGAAGCGGCTGGCGGCGTTGCCAAAGAGTTCAACACCATTGCGGTGGATGATGGGATTGCCATGGGCCACGGGGGGATGCTTTATTCACTGCCATCTCGCGAACTGATCGCTGATTCCGTTGAGTATATGGTCAACGCCCACTGCGCCGATGCCATGGTCTGTATCTCCAACTGCGACAAAATCACCCCTGGGATGTTGATGGCTTCCCTGCGATTAAATATTCCGGTGATCTTTGTTTCCGGCGGCCCGATGGAAGCCGGGAAAACCAAGCTGTCCGATCGGATAATCAAGCTCGATCTGGTTGATGCGATGATCCAGGGCGCAGACCCGAAAGTCTCTGACTCCCAGAGCGATCAGGTTGAACGTTCCGCCTGCCCAACCTGCGGTTCCTGCTCCGGGATGTTTACCGCTAACTCAATGAACTGCCTGACCGAAGCGCTGGGTTTGTCGCAGCCAGGCAACGGCTCGCTGCTGGCAACCCACGCGGACCGTAAGCAGCTGTTCCTTAATGCTGGTAAACGCATTGTTGAATTGACCAAACGTTATTACGAGCAAAACGACGAAAGTGCACTGCCGCGTAATATCGCCAGTAAGGCGGCGTTTGAAAAC GACGCGATAAATCGAAACTGGGGGGTTAA ; SEQ ID NO. 4.

[0034] Gene encoding the dihydroxy acid dehydratase mutant with mutation site C195M ilvD C195M The nucleotide sequence is shown as SEQ ID NO. 5.

[0035] ATGCCTAAGTACCGTTCCGCC ACCACCACCCATGGCCGTAATATGGCGGGGGCCCGCGCACTGTGGCGCGCCACCGGGATGACCGACGCCGATTTCGGTAAGCCGATTATCGCGGTTGTGAACTCGTTCACCCAATTTGTACCGGGTCACGTCCATCTGCGCGATCTCGGTAAACTGGTCGCCGAACAAATTGAAGCGGCTGGCGGCGTTGCCAAAGAGTTCAACACCATTGCGGTGGATGATGGGATTGCCATGGGCCACGGGGGGATGCTTTATTCACTGCCATCTCGCGAACTGATCGCTGATTCCGTTGAGTATATGGTCAACGCCCACTGCGCCGATGCCATGGTCTGTATCTCCAACTGCGACAAAATCACCCCTGGGATGTTGATGGCTTCCCTGCGATTAAATATTCCGGTGATCTTTGTTTCCGGCGGCCCGATGGAAGCCGGGAAAACCAAGCTGTCCGATCGGATAATCAAGCTCGATCTGGTTGATGCGATGATCCAGGGCGCAGACCCGAAAGTCTCTGACTCCCAGAGCGATCAGGTTGAACGTTCCGCCTGCCCAACCTGCGGTTCC ATGGCGATAAATCGAAACTGGGGGGTTAA ; SEQ ID NO. 5.

[0036] Example 1 Obtaining of dihydroxy acid dehydratase gene fragment (1) According to the known dihydroxy acid dehydratase in E. coli with amino acid sequence of SEQ ID NO. 1, which is referred to as wild type in this embodiment, the nucleotide sequence is shown as SEQ ID NO. 3. The key amino acid sites that may affect its biological function are predicted by bioinformatics, and the amino acid sites are mutated to obtain dihydroxy acid dehydratase mutants.

[0037] The site-directed mutagenesis of amino acid is carried out by PCR, specifically by changing the base sequence of the amino acid codon position of the wild type dihydroxy acid dehydratase nucleotide mutant to obtain the gene of dihydroxy acid dehydratase mutant. The nucleotide sequence of the dihydroxy acid dehydratase mutant gene ilvD A268D with mutation site A268D is shown as SEQ ID NO. 4, and the nucleotide sequence of the dihydroxy acid dehydratase mutant gene ilvD C195M with mutation site C195M is shown as SEQ ID NO. 5. Specifically as follows: The wild type dihydroxy acid dehydratase nucleotide sequence (shown as SEQ ID NO. 3) is used as a template, and primers TB-1 and TB-2 are used for amplification to obtain the upstream fragment of the dihydroxy acid dehydratase mutant gene ilvD A268D with mutation site A268D, and primers TB-3 and TB-4 are used for amplification to obtain the downstream fragment of the dihydroxy acid dehydratase mutant gene ilvD A268D with mutation site A268D. The upstream and downstream fragments of the dihydroxy acid dehydratase mutant gene ilvD A268D are subjected to PCR overlap using primers TB-1 and TB-4, and the PCR product is purified and recovered (the product purification kit is purchased from Nanjing Nvwayzhan Biotechnology Co., Ltd.) to obtain the gene fragment of the dihydroxy acid dehydratase mutant with mutation site A268D (shown as SEQ ID NO. 4).

[0038] The wild type dihydroxy acid dehydratase nucleotide sequence (shown as SEQ ID NO. 3) is used as a template, and primers TB-1 and TB-5 are used for amplification to obtain the upstream fragment of the dihydroxy acid dehydratase mutant gene ilvD C195M with mutation site C195M, and primers TB-6 and TB-4 are used for amplification to obtain the downstream fragment of the dihydroxy acid dehydratase mutant gene ilvD C195Mdownstream fragment of the gene ilvD encoding dihydroxy-acid dehydratase mutant with mutation site C195M. The primer TB-1 and TB-4 were used to amplify the gene ilvD encoding dihydroxy-acid dehydratase mutant with mutation site C195M C195M PCR overlap was performed using the upstream and downstream fragments of the gene ilvD encoding dihydroxy-acid dehydratase mutant with mutation site C195M, and the PCR product was recovered after purification (the product purification kit was purchased from Nanjing Nvigan Biotech Co., Ltd.). The gene fragment encoding dihydroxy-acid dehydratase mutant with mutation site C195M was obtained (as shown in SEQ ID NO. 5).

[0039] (2) The nucleotide sequence with His tag (as shown in SEQ ID NO. 2) was used as a template, and the primer His-F and His-R were used to amplify the nucleotide sequence with His tag. The PCR product was recovered after purification (the product purification kit was purchased from Nanjing Nvigan Biotech Co., Ltd.). The gene fragment of the nucleotide sequence with His tag was obtained. The primers TB-1 and TB-4 were used to amplify the wild-type dihydroxy-acid dehydratase gene and the dihydroxy-acid dehydratase mutant gene, respectively. The PCR product was recovered after purification (the product purification kit was purchased from Nanjing Nvigan Biotech Co., Ltd.). The gene fragment of the wild-type dihydroxy-acid dehydratase, the gene fragment encoding dihydroxy-acid dehydratase mutant with mutation site A268D, and the gene fragment encoding dihydroxy-acid dehydratase mutant with mutation site C195M were obtained. The gene fragments of the wild-type dihydroxy-acid dehydratase with His tag, the gene fragment encoding dihydroxy-acid dehydratase mutant with mutation site A268D with His tag, and the gene fragment encoding dihydroxy-acid dehydratase mutant with mutation site C195M with His tag were obtained by PCR overlap technology using primers His-F and TB-4.

[0040] The specific primer sequences are as follows: TB-1: 5'-ATGCCTAAGTACCGTTCCGCC-3'; SEQ ID NO. 6.

[0041] TB-2: 5'-CAGCGTCATGTCGTTTTCAAACGC-3'; SEQ ID NO. 7.

[0042] TB-3: 5'-GCGTTTGAAAACGACATGACGCTG-3'; SEQ ID NO. 8.

[0043] TB-4: 5'-TTAACCCCCCAGTTTCGATTTATCGC-3'; SEQ ID NO. 9.

[0044] TB-5: 5'-TCCCGGACATGGAACCGCAG-3'; SEQ ID NO. 10.

[0045] TB-6: 5'-CTGCGGTTCCATGTCCGGGA-3'; SEQ ID NO. 11.

[0046] His-F: 5'-CGCGAAATTAATACGACTCACTATAGGG-3'; SEQ ID NO. 12.

[0047] His-R: 5'-GCGGAACGGTACTTAGGCAT GCGACCCATTTGCTGTCCACCAG -3'; SEQ ID NO. 13.

[0048] PCR reaction system: 5x buffer 10 μL, 2.5mM dNTPs 4 μL, ddH2O 32 μL, 10µM of upstream primer and downstream primer 1 μL each, DNA template 1 μL, pfu enzyme 1 μL.

[0049] PCR reaction program: initial denaturation, 95℃, 2min; denaturation, 95℃, 20s; annealing, 58℃, 30s; extension, 72℃, 30s; cycle number is 29.

[0050] Example 2 Expression and purification of wild-type dihydroxy acid dehydratase (1) Preparation of wild-type dihydroxy acid dehydratase cloning plasmid and expression bacteria The gene fragment of wild-type dihydroxy acid dehydratase with His tag was connected with the linearized pET28a plasmid amplified by primer to obtain a recombinant plasmid, and the recombinant plasmid was transformed into Escherichia coli DH5α (purchased from Nanjing Novozyme Biotech Co., Ltd.). The pET28a-ilvD plasmid was extracted from the positive transformant by using the positive transformant Figure 1), the pET28a-ilvD plasmid was transformed into Escherichia coli BL21 by heat shock transformation method, and the transformation product was transferred to LB medium to obtain the transformed Escherichia coli. Specifically as follows: the pET28a was used as a template, and Bone-F and Bone-R were used as primers to obtain a linearized pET28a plasmid by PCR amplification. The recovered linearized pET28a plasmid and the gene fragment of the wild-type dihydroxy acid dehydratase with a His tag were connected by Gibson clone to obtain a recombinant plasmid pET28a-ilvD; the connection product was transformed into Escherichia coli DH5α to obtain the transformation product. The transformation product was coated on LB solid medium containing 100 μg / mL kanamycin, and incubated at 37°C in an incubator for 12 h, to obtain the transformant; the transformant was inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and incubated at 37°C, 200 rpm for 12 h, and then the plasmid was extracted and sequenced to verify the purpose gene. After verification, the correct one was obtained as the recombinant plasmid. 50 μL of Escherichia coli BL21 competent cells were taken, ice-bathed, 2 μL of recombinant plasmid was added, ice-bathed for 20 min, 42°C heat shock for 45 s, and then ice-bathed for 2 min. 800 μL of LB was added, and the mixture was shaken at 37°C, 200 rpm for 1 h. 100 μL was taken and uniformly coated on the LB plate containing kanamycin, and incubated at 37°C in an incubator overnight to obtain the transformed Escherichia coli.

[0051] The primer sequences used are as follows: Bone-F: 5-ATCGAAACTGGGGGGTTAATGAGATC-3'; SEQ ID NO. 14.

[0052] Bone-R: 5'-TGAGTCGTATTAATTTCGCGGGATCGAGA-3'; SEQ ID NO. 15.

[0053] The Gibson clone connection was configured with a system of 10 μL, including 5 μL of Gibson enzyme and 5 μL of fragments and skeletons (plasmids).

[0054] (2) Culture of wild-type dihydroxy acid dehydratase expression bacteria The correct transformant single colony was inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and incubated at 37°C, 200 rpm for 12 h to obtain the culture solution; the culture solution was inoculated into 1000 mL of LB liquid medium containing 100 μg / mL kanamycin at 1%, and incubated at 37°C, 200 rpm for 12 h to obtain the culture solution. The culture solution was inoculated into 1000 mL of LB liquid medium containing 100 μg / mL kanamycin at 1%, and incubated at 37°C, 200 rpm for 12 h to obtain the culture solution. 600When the concentration was 0.8, IPTG (final concentration 0.2 mM) was added, the temperature was lowered to 16℃, and the culture was continued to shake at 200 rpm for 18 h to obtain the expression bacteria.

[0055] (3) Purification of wild-type dihydroxy acid dehydratase expression bacteria After obtaining the expression bacteria, the wet bacterial cells were collected by centrifugation at 5000 rpm for 10 min, and 20 mL of Tris-HCl buffer a was added to suspend the bacterial cells. The bacterial suspension was then sonicated in a 4°C ice-water bath. The sonication conditions were: power 250W, on for 3 s, off for 4 s, temperature 4°C, and effective disruption time 15 min. After centrifugation at 12000 rpm for 30 min, the supernatant was collected to obtain the crude enzyme solution. The crude enzyme solution of dihydroxy acid dehydratase was placed in ice water, and protamine sulfate and 1,4-dithiothreitol were added to a final concentration of 1.4 mg / mL. After stirring and mixing, the mixture was reacted on ice for 20 min, centrifuged at 12,000 rpm for 15 min, and the supernatant was retained. The supernatant was allowed to flow naturally down a nickel column, allowing the protein to be adsorbed into the nickel column packing. Impurities were eluted with 50 mM imidazole solution, and the operation was repeated 3 times. The target protein adsorbed in the nickel column was eluted with 100 mM imidazole solution, and the elution was repeated 3 times. The filtrate was collected. 1,4-dithiothreitol was added again to the filtrate to a final concentration of 1 mM, and the mixture was concentrated by ultrafiltration. The enzyme was desalted using a Nap column to obtain the target pure enzyme.

[0056] Example 3 Expression and purification of a dihydroxy acid dehydrase mutant with mutation site A268D (1) Preparation of dihydroxy acid dehydrase cloning plasmid with mutation site A268D and expression bacteria A gene fragment of dihydroxy acid dehydratase with a His-tagged mutation site A268D was ligated with a linearized pET28a plasmid obtained by primer amplification to obtain a recombinant plasmid. This recombinant plasmid was then transformed into *E. coli* DH5α (purchased from Nanjing Novizan Biotechnology Co., Ltd.). pET28a-ilvD was extracted from positive transformants. A268D plasmid ( Figure 2 ), pET28a-ilvD A268D The plasmid was transformed into *E. coli* BL21 via heat shock transformation. The transformation product was then transferred to LB medium to obtain transformant *E. coli*. Specifically, using pET28a as a template and Bone-F and Bone-R primers, PCR amplification was performed to obtain the linearized pET28a plasmid. The recovered linearized pET28a plasmid and the gene fragment containing the His-tagged mutation site A268D of dihydroxy acid dehydratase were ligated using Gibson clone to obtain the recombinant plasmid pET28a-ilvD. A268D; The ligation product was transformed into Escherichia coli DH5a to obtain a transformation product. The transformation product was coated on LB solid medium containing 100 μg / mL kanamycin and incubated in a 37 °C constant temperature incubator for 12 h to obtain transformants; the transformants were inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and after 12 h of shake flask culture at 37 °C and 200 rpm, the plasmid was extracted and sequenced, and then aligned with the target gene to verify the correctness. If correct, the recombinant plasmid was obtained. 50 μL of Escherichia coli BL21 competent cells were taken, ice-bathed, 2 μL of recombinant plasmid was added, ice-bathed for 20 min, 42 °C heat shock for 45 s, and then ice-bathed for 2 min. 800 μL of LB was added, and the mixture was shaken at 37 °C and 200 rpm for 1 h. 100 μL was taken and uniformly coated on an LB plate containing kanamycin, and then incubated in a 37 °C incubator overnight to obtain the transformant Escherichia coli.

[0057] (2) Culture of dihydroxy acid dehydratase expression bacteria with mutation site A268D The correct transformant single colony was inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and then incubated at 37 °C and 200 rpm for 12 h to obtain a culture solution; the culture solution was inoculated into 1000 mL of LB liquid medium containing 100 μg / mL kanamycin at a ratio of 1%, and then incubated at 37 °C and 200 rpm until the OD 600 was 0.8. Then, IPTG was added (final concentration 0.2 mM), the temperature was reduced to 16 °C, and the mixture was further incubated at 200 rpm for 18 h to obtain the expression bacteria.

[0058] (3) Purification of dihydroxy acid dehydratase expression bacteria with mutation site A268D After obtaining the expression bacteria, collect the wet bacteria by centrifugation at 5000 rpm for 10 min, and add 20 mL of Tris-HCl buffer a to suspend the bacteria. The bacterial suspension is ultrasonically broken in a low-temperature ice water bath at 4°C, with the ultrasonic breaking conditions being: power 250 W, 3 s on and 4 s off, temperature 4°C, and effective breaking time 15 min. After the ultrasonically broken mixture is centrifuged at 12000 rpm for 30 min, the supernatant is obtained, that is, the crude enzyme solution. The crude enzyme solution is placed in ice water, and 1.4 mg / mL of protamine sulfate and 2 mM of 1,4-dithiothreitol are added, and after stirring and mixing, the reaction is carried out on ice for 20 min, and centrifugation is carried out at 12000 rpm for 15 min, and the supernatant is reserved; the supernatant is naturally flowed in a nickel column, so that the protein is adsorbed on the filler of the nickel column, and 50 mM of imidazole solution is used to elute the impurities, and the operation is repeated for 3 times; 100 mM of imidazole solution is used to elute the target protein adsorbed in the nickel column, and the elution is carried out for 3 times, and the filtrate is collected; 1 mM of 1,4-dithiothreitol is added to the filtrate again, and ultrafiltration and concentration are carried out; desalting is carried out on a Nap column, and the target pure enzyme is obtained.

[0059] Example 4 Expression and purification of dihydroxy acid dehydratase mutant with mutation site C195M (1) Preparation of dihydroxy acid dehydratase mutant plasmid and expression bacteria with mutation site C195M The gene fragment of dihydroxy acid dehydratase with mutation site C195M and the linearized pET28a plasmid amplified by the primer are connected to obtain a recombinant plasmid, and the recombinant plasmid is transformed into Escherichia coli DH5α (purchased from Nanjing Novozyme Bio-tech Co., Ltd.). The positive transformant is used to extract pET28a-ilvD C195M plasmid. Figure 3 The pET28a-ilvD C195M plasmid is transformed into Escherichia coli BL21 by heat shock transformation, and the transformation product is transferred to LB medium to obtain the transformant Escherichia coli. Specifically, the linearized pET28a plasmid is obtained by PCR amplification with pET28a as the template and Bone-F and Bone-R as the primers. The recovered linearized pET28a plasmid and the gene fragment of dihydroxy acid dehydratase with mutation site C195M are connected by Gibson clone to obtain a recombinant plasmid pET28a-ilvD C195M; The ligation product was transformed into Escherichia coli DH5a to obtain a transformation product. The transformation product was coated on LB solid medium containing 100 μg / mL kanamycin and incubated in a 37 °C constant temperature incubator for 12 h to obtain transformants; the transformants were inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and after 12 h of shake flask culture at 37 °C and 200 rpm, the plasmid was extracted and sequenced, and then aligned with the target gene to verify the correctness. If correct, the recombinant plasmid was obtained. 50 μL of Escherichia coli BL21 competent cells were taken, ice-bathed, 2 μL of recombinant plasmid was added, ice-bathed for 20 min, 42 °C heat shock for 45 s, and then ice-bathed for 2 min. 800 μL of LB was added, and the mixture was shaken at 37 °C and 200 rpm for 1 h. 100 μL was taken and uniformly coated on an LB plate containing kanamycin, and then incubated in a 37 °C incubator overnight to obtain the transformant Escherichia coli.

[0060] (2) Culture of dihydroxy acid dehydratase expression bacteria with mutation site C195M The correct transformant single colony was inoculated into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and then incubated at 37 °C and 200 rpm for 12 h to obtain a culture solution; the culture solution was inoculated into 1000 mL of LB liquid medium containing 100 μg / mL kanamycin at a ratio of 1%, and then incubated at 37 °C and 200 rpm until the OD 600 was 0.8. Then, IPTG was added (final concentration 0.2 mM), the temperature was reduced to 16 °C, and the incubation was continued at 200 rpm for 18 h to obtain the expression bacteria.

[0061] (3) Purification of dihydroxy acid dehydratase expression bacteria with mutation site C195M After obtaining the expression bacteria, centrifuge at 5000 rpm for 10 min to collect the wet bacteria, and add 20 mL of Tris-HCl buffer a to suspend the bacteria. The bacterial suspension is ultrasonically broken in a low-temperature ice water bath at 4°C, and the ultrasonic breaking conditions are: power 250W, 3s on and 4s off, temperature 4°C, and effective breaking time 15 min. After the ultrasonic breaking mixture is centrifuged at 12000 rpm for 30 min, the supernatant is obtained, which is the crude enzyme solution. The crude enzyme solution is placed in ice water, and 1.4 mg / mL of protamine sulfate and 2 mM of 1,4-dithiothreitol are added, stirred and mixed, and then reacted on ice for 20 min. After centrifugation at 12000 rpm for 15 min, the supernatant is reserved. The supernatant is naturally flowed in a nickel column, so that the protein is adsorbed on the filler of the nickel column. The impure protein is eluted with 50 mM of imidazole solution, and the operation is repeated 3 times. The target protein adsorbed in the nickel column is eluted with 100 mM of imidazole solution, and the elution is repeated 3 times. The filtrate is collected. 1 mM of 1,4-dithiothreitol is added to the filtrate, and the filtrate is concentrated by ultrafiltration. The Nap column is desalted to obtain the target pure enzyme.

[0062] Example 5 Enzyme activity determination method of dihydroxy acid dehydratase 10 mL of pyruvic acid reaction solution is added with 100 µg of acetolactate synthase to catalyze pyruvic acid to synthesize 2-acetyllactic acid, and the reaction time is 10 min. Further, 100 µg of ketol-acid reductoisomerase and 1 mL of 0.1 mM NAD(P)H solution are added to catalyze 2-acetyllactic acid to synthesize 2,3-dihydroxyisovaleric acid, and the reaction time is 10 min. The final solution obtained is the substrate solution. In 9.2 mL of the substrate solution, 100 µg of dihydroxy acid dehydratase is added, 0.8 mL of the reconstituted iron-sulfur cluster solution is added, and the reaction is carried out for 10 min to synthesize α-ketoisovalerate (α-KIV) (see Table 1). Figure 4 ), 1 mL of 2M hydrochloric acid solution is added to terminate the reaction, and the enzyme reaction solution test sample solution is obtained. In 9.2 mL of the substrate solution, 0.8 mL of the reconstituted iron-sulfur cluster solution is added, mixed, and then placed for 10 min. Then, 1 mL of 2M hydrochloric acid solution is added, and the blank control solution is obtained. The standard curve is established by high performance liquid chromatography with the concentration gradient of α-ketoisovalerate standard as the abscissa and the peak area as the ordinate. The test sample solution and the blank control solution are analyzed, and the enzyme activity is calculated. The results are shown in Table 1.

[0063] The standard curve is y=ax+b=14312x-49.008 (see Table 1). Figure 5 ).

[0064] The enzyme activity calculation formula is: Enzyme activity = [(A0-A1) x N x V] / [m x t x M x a]. Wherein a is the slope of the standard curve, b is the intercept of the standard curve; A0is the peak area of a-ketoisovalerate in the blank control solution; A1is the peak area of a-ketoisovalerate in the solution after the reaction; V is the total volume of the reaction system; m is the mass of dihydroxy acid dehydratase sample; t is the reaction time of enzyme and substrate; M is the molar mass of a-ketoisovalerate; N is the dilution multiple of enzyme solution to test solution.

[0065] 50 mM imidazole solution is 0.34 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with concentrated hydrochloric acid (12 M).

[0066] 100 mM imidazole solution is 0.68 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with concentrated hydrochloric acid (12 M).

[0067] Pyruvate reaction solution is 10 mM pyruvate, 2 mM thiamine pyrophosphate, 20 mM MgCl2, and solvent is Tris-HCl buffer c.

[0068] Reconstituted iron-sulfur cluster solution is 20 mM MgCl2, 20 mM FeSO4, 20 mM L-cysteine, 2 mM 1,4-dithiothreitol, and solvent is ultrapure water.

[0069] High performance liquid chromatography conditions are: mobile phase 0.5 mM sulfuric acid, mobile phase flow rate is 0.6 mL / min; detection wavelength is 340 nm.

[0070] Tris-HCl buffer a is 50 mM Tris reagent, 10 mM MgCl2, solvent is ultrapure water, and the pH is adjusted to 8.0 with concentrated hydrochloric acid (12 M).

[0071] Tris-HCl buffer b is 50 mM Tris reagent, 150 mM NaCl, 10 mM MgCl2, solvent is ultrapure water, and the pH is adjusted to 8.0 with concentrated hydrochloric acid (12 M).

[0072] Tris-HCl buffer c is 50 mM Tris reagent, solvent is ultrapure water, and the pH is adjusted to 8.0 with concentrated hydrochloric acid (12 M).

[0073] Table 1 Enzymatic activity (U / mg) of dihydroxy acid dehydratase

[0074] Table 1 is the determination of effective enzyme activity of dihydroxy acid dehydratase. Sample A is dihydroxy acid dehydratase with amino acid sequence of SEQ ID NO. 1, which is called wild type in this embodiment, and its encoding gene ilvD nucleotide sequence is shown in SEQ ID NO. 3; sample B is dihydroxy acid dehydratase mutant with mutation site A268D, and its encoding gene ilvD nucleotide sequence is shown in SEQ ID NO. 4; sample C is dihydroxy acid dehydratase mutant with mutation site C195M, and its encoding gene ilvD nucleotide sequence is shown in SEQ ID NO. 5. A268D C195M

[0075] The method of the present application can realize the in vitro purification of dihydroxy acid dehydratase by in vitro reconstitution of iron-sulfur cluster purification method, without the need for additional control of oxygen, and can maintain the stability of the catalytic properties of dihydroxy acid dehydratase. In addition, the enzyme activity is successfully determined by a three-step cascade reaction using conventional reagents instead of expensive substrates, thereby realizing the determination of enzyme activity at low cost.

[0076] The above description of disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.​​

Claims

1. A method for purifying and assaying the enzyme activity of a dihydroxy-acid dehydratase, characterized by, Comprising the following steps: (1) Purification of dihydroxy acid dehydratase Put the crude enzyme solution of dihydroxy acid dehydratase in ice water, add protamine sulfate with a final concentration of 1.0-1.4 mg / mL and 1,4-dithiothreitol with a final concentration of 0.5-2 mM, stir evenly, react on ice for 10-20 min, centrifuge at 12000 rpm for 10-15 min, and reserve the supernatant; let the supernatant flow naturally in a nickel column, so that the protein is adsorbed on the filler of the nickel column, use 50 mM imidazole solution to elute the impurities, repeat the operation 1-3 times; use 100 mM imidazole solution to elute the target protein adsorbed in the nickel column, elute 2-3 times, and collect the filtrate; add 1,4-dithiothreitol with a final concentration of 0.1-1 mM to the filtrate again, and concentrate by ultrafiltration; desalt with a Nap column to obtain the target pure enzyme; (2) Enzyme activity determination of dihydroxy acid dehydratase Add 100 µg of acetolactate synthase to 10 mL of pyruvic acid reaction solution, and the reaction time is 10 min; after the reaction is completed, add 100 µg of ketonic acid reduction isomerase and 1 mL of 0.5-1 mM NAD(P)H solution, and the reaction time is 10 min to obtain a substrate solution; add 100 µg of dihydroxy acid dehydratase to 9.2 mL of the substrate solution, add 0.8 mL of reconstituted iron-sulfur cluster solution, and react for 10 min to synthesize α-ketoisovaleric acid; add 1 mL of 2M hydrochloric acid solution to terminate the reaction, and obtain an enzymatic reaction liquid test solution; add 0.8 mL of reconstituted iron-sulfur cluster solution to 9.2 mL of the substrate solution, mix evenly, stand for 10 min, and then add 1 mL of 2M hydrochloric acid solution to obtain a blank control solution; The pyruvic acid reaction solution is 10-20 mM pyruvic acid, 0.5-2 mM thiamine pyrophosphate, 10-20 mM MgCl2, and the solvent is Tris-HCl buffer c; The reconstituted iron-sulfur cluster solution is 10-20 mM MgCl2, 10-20 mM FeSO4, 10-20 mM L-cysteine, and 0.5-2 mM 1,4-dithiothreitol, and the solvent is ultrapure water; A standard curve is established by high-performance liquid chromatography with the concentration gradient of α-ketoisovaleric acid standard as the abscissa and the peak area as the ordinate, and the test solution and the blank control solution are analyzed to calculate the enzyme activity; The standard curve is y=ax+b=14312x-49.008; The enzyme activity calculation formula is: Enzyme activity=[(A0- A1) x N x V] / [m x t x M x a]; Wherein a is the slope of the standard curve, b is the intercept of the standard curve; A0 is the peak area of α-ketoisovaleric acid in the blank control solution; A1 is the peak area of α-ketoisovaleric acid in the solution after the reaction is completed; V is the total volume of the reaction system; m is the mass of the dihydroxy acid dehydratase sample; t is the reaction time of the enzyme and the substrate; M is the molar mass of α-ketoisovaleric acid; N is the dilution multiple of the enzyme solution to the test solution.

2. The method for purification and determination of enzyme activity of dihydroxy-acid dehydratase according to claim 1, wherein, The dihydroxy acid dehydratase in step (1) is a wild type and a mutant of dihydroxy acid dehydratase; The mutant of dihydroxy acid dehydratase is A268D or C195M; The amino acid sequence of the wild type of dihydroxy acid dehydratase is shown in SEQ ID NO.

1.

3. The method for purification and determination of enzyme activity of dihydroxy-acid dehydratase according to claim 2, wherein, The preparation method of the crude enzyme solution of dihydroxy acid dehydratase in step (1) is as follows: The recombinant plasmid obtained by linking the gene fragment of His-tagged dihydroxy acid dehydratase with the vector pET28a was transformed into E. coli BL21, and the correct transformants were cultured to OD 600 When OD600 was 0.6-0.8, 0.05-0.2 mM IPTG was added to induce expression at 16°C for 12-18 h, and then the cells were resuspended in 15-20 mL of Tris-HCl buffer a, and then the cells were broken, centrifuged, and the supernatant was obtained to obtain the crude enzyme solution; The nucleotide sequence with His tag is shown in SEQ ID NO. 2; and the gene fragment of dihydroxy acid dehydratase is shown in any one of SEQ ID NO. 3-5.

4. The method for purifying and determining the activity of dihydroxy acid dehydratase according to claim 3, wherein, The 50 mM imidazole solution is 0.34 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The 100 mM imidazole solution is 0.68 g of imidazole dissolved in 100 mL of Tris-HCl buffer b, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer a is 50 mM Tris reagent, 10-30 mM MgCl2, solvent is ultrapure water, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer b is 50 mM Tris reagent, 150 mM NaCl, 10-30 mM MgCl2, solvent is ultrapure water, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid; The Tris-HCl buffer c is 50 mM Tris reagent, solvent is ultrapure water, and the pH is adjusted to 8.0 with 12M concentrated hydrochloric acid.

5. The method for purifying and determining the activity of dihydroxy acid dehydratase according to claim 4, wherein, The high performance liquid chromatography condition in step (2) is that the mobile phase is 0.5 mM sulfuric acid, the flow rate of the mobile phase is 0.3-0.8 mL / min, and the detection wavelength is 315-340 nm.

Images