CN117402851A - Polyglucose kinase mutant and application thereof - Google Patents

Polyglucose kinase mutant and application thereof Download PDF

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CN117402851A
CN117402851A CN202311176882.2A CN202311176882A CN117402851A CN 117402851 A CN117402851 A CN 117402851A CN 202311176882 A CN202311176882 A CN 202311176882A CN 117402851 A CN117402851 A CN 117402851A
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mutant
kinase
polyglucose
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arabinose
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薛洪泽
侯伟宏
魏顺新
牛继如
马向辉
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Tianjin Famoxi Biomedical Technology Co ltd
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Abstract

The invention belongs to the field of synthetic biology, and provides a polyglucose kinase mutant and application thereof, wherein the amino acid sequence of the mutant is the amino acid sequence with mutation shown in SEQ ID NO. 2, and the mutated amino acid are L102V, N Q, E150T and H153E, G N. A mutant of polyglucose kinase having at least one mutation site or retaining the mutation site and having more than 90% amino acid sequence homology with the mutant, wherein an enzyme-catalyzed optimal substrate is changed from D-glucose to D-arabinose. The 5-phosphate-D-arabinose can be further used for preparing compounds such as arabinoside, cytarabine, ara-adenosine, 2-amino ara-adenosine, ara-guanosine and the like.

Description

Polyglucose kinase mutant and application thereof
Technical Field
The invention belongs to the field of synthetic biology, and particularly relates to a polyglucose kinase mutant and application thereof.
Background
Nucleosides are a class of polyhydroxy compounds that are precursors to natural nucleic acids, as well as structural components of many coenzymes. Therefore, the nucleoside and the derivative thereof have important application value in the fields of medicine, biochemistry and biotechnology. At present, nucleoside compounds play an important role in anti-AIDS, anti-tumor and other viral diseases treatment drugs applied clinically. Many nucleoside analogues are similar in structure to naturally occurring nucleosides and cannot be recognized by viruses in vivo, so that the nucleoside analogues can be effectively embedded in viral DNA chains, and can be competitively doped with nucleotides into the viral DNA chains to inhibit enzymes in the viral replication process, thereby inhibiting replication and reverse transcription of viral DNA, stopping or inhibiting extension and synthesis of viral DNA chains, and interfering expression of viral genes, so that the aim of treating diseases is fulfilled.
The preparation methods of nucleoside compounds and derivatives thereof can be divided into two types, namely chemical methods and enzymatic methods. Chemical methods are the traditional and mainstream methods of preparing such drugs at present. In recent years, the enzymatic preparation of nucleoside analogues has the advantages of high efficiency, high regioselectivity, mild reaction conditions, simplicity, controllability, environmental friendliness and the like, and overcomes the defects of low selectivity, more byproducts, more preparation and purification steps and the like of the common chemical method, so that the nucleoside analogues are increasingly valued. With the development of biotechnology, enzyme is used as a catalyst to link bases and ribose, which is a more common method in the field of preparation of nucleoside substances. When the nucleoside compound is synthesized by enzyme catalysis, the complex protection and deprotection steps are not needed for the base and ribose, so that the method has great economic and social benefits.
Arabinonucleosides are one type of nucleoside analogues, mainly consisting of arabinose linked to the base instead of ribose. Such compounds have been widely used in the pharmaceutical field, for example fludarabine, nelarabine, cytarabine, and the like. The synthesis of the compounds generally adopts relatively easily available arabinonucleoside (generally arabinoside, shown in formula b) as a raw material, and the corresponding products are obtained through two-step enzyme catalytic synthesis. As reported by Li Ping et al (Biotechnol bioeng.2022, 119, 1768-1780), expression of uridine phosphorylase and purine nucleoside phosphorylase in E.coli catalyzes the synthesis of cytarabine from arabinoside and cytosine (formula c). Also, as reported in the chinese patent application publication No. CN106929553a, the synthesis of arabinoside (formula d) from arabinoside and adenine by co-catalysis of uridine phosphorylase and purine nucleoside phosphorylase is reported. Furthermore, according to Liu Guosheng et al (J.Chi.A., 2016, volume 47, 857-860), it has been reported that arabino-guanosine (formula f) is produced from 2, 6-diaminopurine nucleoside (formula e) as a raw material by whole cell catalysis of Pseudomonas aeruginosa. However, the main starting material used in the above-described process is arabinonucleoside, which is hydrolysed to the base and 1-phospho-D-arabinose by a first enzyme, and subsequently the second enzyme catalyzes the synthesis of the corresponding nucleoside product from 1-phospho-D-arabinose and further base. The product is mainly obtained by base exchange through two steps of reactions, the price of raw materials is relatively high, and the product yield is relatively low.
Another method for obtaining 1-phosphate-D-arabinose is to catalyze the 5-hydroxyl phosphorylation of D-arabinose by ribose kinase or xylose kinase, but the phosphate donor of the enzyme is ATP, and the price is still relatively high. Although ATP can be regenerated in other ways, many regeneration methods face thermodynamic equilibrium problems and raw material costs (J. Jarooensuk et al, molecular Catalysis,2023,537,112937).
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a polyphosphate glucokinase mutant and application thereof, wherein the polyphosphate glucokinase mutant has high catalytic activity and good thermal stability on D-arabinose, and the enzyme is matched with different nucleoside phosphorylases to prepare the arabinoside, the 2-amino arabinoside and the arabinoside.
The technical scheme adopted by the invention is as follows: a mutant of the polyphosphoric glucokinase has an amino acid sequence shown in SEQ ID NO 2.
The invention modifies enzyme protein by combining rational design, directed evolution and high-throughput screening technology of enzyme. During the engineering process, the inventors found that the polyglucose kinase from Chloracidobacterium thermophilum was found in L102V, N Q, E150T, H153E, G205N. After mutation, the catalytic activity of the enzyme on arabinose is obviously improved. The amino acid sequence of the polyglucose kinase mutant is SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2. The amino acid sequence of the mutant phosphoglucose kinase has mutation sites in the mutated amino acid sequence and has more than 90% of homology with the mutated amino acid sequence.
A gene of a mutant of the polyphosphate glucokinase, which is derived from Chloracidobacterium thermophilum wild type polyphosphate glucokinase SEQ ID NO 1. The substrate for the enzyme obtained after expression of these genes is glucose, while the catalytic activity for D-arabinose is low. The invention provides a polyphosphoric acid glucokinase mutant which has higher activity to a substrate shown in a formula a.
The technical scheme adopted by the invention is as follows: a recombinant carrier containing the gene of the polyphosphoric acid glucokinase mutant is constructed by connecting the nucleotide sequence of the polyphosphoric acid glucokinase gene to various prokaryotic expression carriers or eukaryotic expression carriers, such as pGEX, pMAL, pET series prokaryotic expression carriers and eukaryotic expression carriers. Preferably, pET series plasmids are used as starting vectors, in particular pET-24a.
The technical scheme adopted by the invention is as follows: a genetically engineered bacterium for producing the mutant of the polyglucose kinase comprises the recombinant vector, and the host cell is escherichia coli (Escherichia coli BL (DE 3)).
The technical scheme adopted by the invention is as follows: a method for preparing a mutant of polyglucose kinase, comprising the following steps: culturing the genetically engineered bacteria to obtain recombinant polyglucose kinase mutant.
Further, the method comprises the steps of fermenting and culturing the genetically engineered bacteria, and collecting and preparing recombinant polyglucose kinase mutants. Specifically, the step of industrially preparing the recombinant polyglucose kinase and mutants thereof is carried out in a fermentation tank according to certain fermentation conditions; the fermentation conditions of the production tank are preferably as follows: DO is above 20%, and the air flow is 1:0.5-2 vvm.
The technical scheme adopted by the invention is as follows: the application of the polyphosphate glucokinase mutant is applied to a phosphate transfer reaction, takes the polyphosphate as a phosphate donor, phosphorylates D-arabinose shown in a formula a, and is further used for preparing beta-D-arabinonucleoside shown in a formula b, a formula c, a formula D, a formula e and a formula f;
formula a is D-arabinose, formula b is arabinoside, formula c is cytarabine, formula D is arabinoside, formula e is 2-amino arabinoside, and formula f is arabinogaoside.
The invention uses low-price and easily available D-arabinose and polyphosphoric acid as raw materials to produce 5-phosphoric acid-D-arabinose, and then uses glucose phosphate isomerase to catalyze and synthesize 1-phosphoric acid-D-arabinose. Most of the studies have been carried out with the phosphate donor ATP being very expensive in the preparation of 5-phosphate-arabinose. The polyphosphate dependent glucokinase mutant adopted by the invention takes the polyphosphate as a phosphate donor, but most of the enzymes take glucose as a substrate, the catalytic activity is low when catalyzing the phosphorylation of the arabinose, and the invention combines the rational design, directed evolution and high-flux screening technology of the enzyme to modify the enzyme protein, so as to obtain the polyphosphate glucokinase mutant with high D-arabinose catalytic activity and good thermal stability. The invention combines the mutant of the polyphosphoric glucokinase and isomerase, pyrimidine nucleoside phosphorylase or purine nucleoside phosphorylase to synthesize the arabinoside, the cytarabine, the arabinoside, the 2-amino arabinoside and the arabino-guanosine.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a mutant of polyphosphate glucokinase, which can improve the activity of enzyme on a compound shown in a formula a, and can be used for the synthesis reaction of arabinoside with structures shown in a formula b, a formula c, a formula d, a formula e and a formula f.
2. The enzyme has excellent catalytic activity on D-arabinose; when the mutant is used for preparing the compounds shown in the formulas b, c, D, e and f, the adopted raw materials are D-arabinose and polyphosphoric acid which are easier to obtain and have low cost; when the arabinonucleoside is prepared, the catalyzed reaction is simple and mild, no waste is discharged, the reaction conversion rate is high, and the method has a good application prospect.
Detailed Description
In order to enable those skilled in the art to better understand the technical scheme of the present invention, the present invention will be described in detail with reference to specific embodiments.
The embodiment of the invention provides a polyglucose kinase mutant and application thereof, which specifically comprises the following steps:
1: acquisition of thermostable polyglucose kinase
The wild type polyglucose kinase shown in SEQ ID No.1 is subjected to DNA codon optimization, then a gene synthesis company artificially synthesizes a complete gene fragment, the gene is inserted into NdeI and BamHI sites of pET-24a plasmid, and the connected vector is transferred into escherichia coli BL21 (DE 3) to establish the polyglucose kinase genetic engineering strain.
2: mutant of polyphosphoric acid glucokinase
The three-dimensional structure of the polyphosphate glucokinase (SEQ ID NO 1) derived from Chloracidobacterium thermophilum has not been revealed yet. However, the invention uses SWISS-MODEL to construct a three-dimensional MODEL of the wild-type gene sequence, and discovers that the homology of the wild-type gene sequence with uracil nucleoside phosphorylase (PDB ID:1 woq) from Arthrobacter sp.strain KM is 44.44%, and the enzyme functions are similar. Thus, by referring to the three-dimensional structure of the enzyme, the binding simulation of the substrate of formula a to the protein was performed by molecular docking software, and finally by Pymol analysis, the amino acids that are likely to be associated with D-arabinose binding were selected to be 65P, 66S, 78N, 104N, 105D, 106A, 135I, 136G, 150E, 153H, 162E, the sites that bind to ATP, ADP or polyphosphoric acid included 6G, 7G, 8T, 9G, 11K, 28R, 132G, 133T, 167L, 205G, 230N, and the amino acids of these sites were taken as rationally designed mutation sites for saturation mutation.
In addition to the rational design, the invention utilizes an error-prone PCR random mutation method to carry out protein engineering modification on the polyglucose kinase. In general, error-prone PCR can be used to randomly introduce mutations into a target gene at a certain frequency by adjusting reaction conditions (e.g., increasing magnesium ion concentration, adding manganese ions, changing dNTP concentrations of four species in a system, or using low-fidelity DNA polymerase) to change the mutation frequency during amplification when the target gene is amplified by DNA polymerase, thereby obtaining random mutants of protein molecules.
The invention adopts Taq polymerase with lower fidelity and simultaneously utilizes Mn 2+ Substitution of the natural cofactor Mg 2+ Increasing the probability of error.
The 50. Mu.L PCR system was as follows:
wherein: the polyglucose kinase template gene is recombinant plasmid constructed by inserting a gene with SEQ ID NO.1 and optimized codons into pET-24a plasmid; the invention designs the primer according to the upstream and downstream sequences of the target gene in the recombinant plasmid.
The PCR reaction conditions were: pre-denaturation at 95℃for 2.5min; denaturation at 94℃for 15s, annealing at 53℃for 30s, extension at 72℃for 60s for 35 cycles; the extension was continued at 72℃for 10min and cooled to 4 ℃.
The PCR amplified product was ligated to pET-24a vector and transferred into E.coli BL21 (DE 3) to create a polyglucose kinase gene mutation library.
The Escherichia coli BL21 (DE 3) is used as a host, and the pET-24a plasmid is used as a vector to express the polyphosphate glucokinase. The amino acid sequence of the screened high-activity polyglucose kinase mutant gene is shown as SEQ ID NO 2.
3: small-scale production of polyglucokinase and its mutant, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase in shake flask
Coli containing the recombinant plasmid was inoculated into 50mL of LB medium (peptone 10g/L, yeast extract 5g/L, naCl 10g/L, pH 7.2) containing kanamycin (50. Mu.g/mL). Shake-culturing at 37℃and 210rpm for 16 hours. Then transferring into 100mL LB medium containing kanamycin according to the ratio of 1:100, shake culturing at 37deg.C and 210rpm, and measuring absorbance (OD) of bacterial liquid at 600nm at regular time 600 ) To monitor the cell growth density. When the OD of the culture 600 When the enzyme is in the range of 0.6 to 0.8, isopropyl beta-D-thiogalactoside (IPTG) with the final concentration of 0.2mM is added to induce the expression of the target enzyme gene, and the target enzyme gene is induced and cultured overnight (more than or equal to 16 hours). Centrifugation at 10000rpm at 4℃for 10min, discarding the supernatant, and after re-suspending the cell pellet at 200g/L with pre-chilled 100mM phosphate buffer (pH 7.0), ultrasonication, centrifugation at 13000rpm at 4℃for 30min, collecting the supernatant, i.e., crude enzyme solution, and storing at-20 ℃.
4: preparation of cytarabine
The preparation system of cytarabine is as follows:
4mM magnesium sulfate, 200mM D-glucose, 100mM polyphosphate, 50mM HEPES buffer, 30mM cytosine, 100ml/L of wild-type polyglucose kinase or mutant crude enzyme solution thereof, 100ml/L of Thermococcus kodakarensis phosphoglucose isomerase (SEQ ID NO. 3), 100ml/L of Geobacillus stearothermophilus pyrimidine nucleoside phosphorylase (SEQ ID NO. 4) crude enzyme solution, and pH=7.5 of the system was adjusted with sodium hydroxide. The reaction was carried out at 55℃for 3h. When wild-type polyglucose kinase was used as the catalytic enzyme, the yield was 9% in terms of cytosine. When the mutant of polyglucose kinase is used as the catalytic enzyme, the yield is 69% calculated by cytosine.
5: preparation of Alzhugan
4mM magnesium sulfate, 200mM D-glucose, 100mM polyphosphate, 50mM HEPES buffer, uracil 30mM, wild-type polyglucose kinase or its mutant crude enzyme solution 100ml/L, thermococcus kodakarensis phosphoglucose isomerase (SEQ ID NO. 3) 100ml/L, shewanella oneidensis uridine phosphorylase (SEQ ID NO. 5) crude enzyme solution 100ml/L, and the system was adjusted to pH=7.5 with sodium hydroxide. The reaction was carried out at 55℃for 3h. When wild-type polyglucose kinase was used as the catalytic enzyme, the yield was 12% in terms of uracil. When the mutant of polyglucose kinase is used as the catalytic enzyme, the yield is 76% based on uracil.
6: preparation of arabinoside
4mM magnesium sulfate, 200mM D-glucose, 100mM polyphosphate, 50mM HEPES buffer, adenine 30mM, crude enzyme solution of wild type polyglucokinase or mutant thereof 100ml/L, crude enzyme solution of Thermococcus kodakarensis phosphoglucose isomerase (SEQ ID NO. 3) 100ml/L, aeropyrumpernix purine nucleoside phosphorylase (SEQ ID NO. 6) 100ml/L, and pH of the system adjusted with sodium hydroxide=7.5. The reaction was carried out at 55℃for 3h. When wild-type polyglucose kinase was used as the catalytic enzyme, the yield was 13% in terms of adenine. When the mutant of polyglucose kinase is used as the catalytic enzyme, the yield is 89% in terms of adenine.
7: preparation of 2-amino arabinoside
4mM magnesium sulfate, 200mM D-glucose, 100mM polyphosphate, 50mM HEPES buffer, 30mM 2-aminoadenine, 100ml/L of wild-type polyglucose kinase or mutant crude enzyme solution, 100ml/L, thermoclostridiumcaenicola purine nucleoside phosphorylase (SEQ ID NO. 3) 100ml/L of Thermococcus kodakarensis phosphoglucose isomerase (SEQ ID NO. 7) crude enzyme solution, and the system was adjusted to pH=7.5 with sodium hydroxide. The reaction was carried out at 55℃for 3h. When wild-type polyglucose kinase was used as the catalytic enzyme, the yield was 11% based on 2-aminoadenine. When the mutant of polyglucose kinase is used as the catalytic enzyme, the yield is 86% based on 2-amino adenine.
8: preparation of arabinogalactan
4mM magnesium sulfate, 200mM D-glucose, 100mM polyphosphate, 50mM HEPES buffer, guanine 30mM, crude enzyme solution of wild-type polyglucose kinase or mutant thereof 100ml/L, crude enzyme solution of Thermococcus kodakarensis phosphoglucose isomerase (SEQ ID NO. 3) 100ml/L, halomonas elongata purine nucleoside phosphorylase (SEQ ID NO. 8) 100ml/L, and the system was adjusted in valence pH=7.5 with sodium hydroxide. The reaction was carried out at 55℃for 3h. When wild-type polyglucose kinase was used as the catalytic enzyme, the yield was 11% based on 2-aminoadenine. When the mutant of polyglucose kinase is used as the catalytic enzyme, the yield is 86% based on 2-amino adenine.
8: HPLC detection (yield) of nucleosides
The measurement is carried out by high performance liquid chromatography (China pharmacopoeia 2020 edition, four-part rule 0512).
Solvent: water-methanol (85:15).
Chromatographic conditions: octadecylsilane chemically bonded silica (4.6X105 mm,5 μm or column with equivalent performance) is used as filler; water (0.1% phosphoric acid) is used as a mobile phase A, acetonitrile is used as a mobile phase B, and linear gradient elution is carried out; the flow rate is 1.0ml per minute; the column temperature is 35 ℃; the detection wavelength is 254nm; the sample volume was 5. Mu.l.
Table 1 table of variation of mobile phase ratio in HPLC detection method
Time (minutes) Mobile phase a (%) Mobile phase B (%)
0 90 10
3 90 10
15 60 40
18 10 90
18.1 90 10
22 90 10
Assay: precisely measuring the solution of the sample, injecting into a liquid chromatograph, and recording the chromatogram.
The present invention has been described in detail by way of examples, but the description is merely exemplary of the invention and should not be construed as limiting the scope of the invention. The scope of the invention is defined by the claims. In the technical scheme of the invention, or under the inspired by the technical scheme of the invention, similar technical schemes are designed to achieve the technical effects, or equivalent changes and improvements to the application scope are still included in the protection scope of the patent coverage of the invention.

Claims (6)

1. A mutant polyphosphate glucokinase, characterized in that: the amino acid sequence is shown as SEQ ID NO 2.
2. A recombinant vector comprising the gene of the mutant polyglucose kinase of claim 1, characterized in that: pET series plasmid is taken as an initial vector.
3. A genetically engineered bacterium for producing the polyglucose kinase mutant of claim 1, characterized in that: the genetically engineered bacterium comprises the recombinant vector of claim 2, and the host cell is escherichia coli.
4. A method for preparing a mutant of polyglucose kinase, which is characterized by comprising the following steps: culturing the genetically engineered bacterium of claim 3 to obtain a recombinant polyglucose kinase mutant.
5. The method for producing a mutant of polyglucose kinase as defined in claim 4, wherein: fermenting and culturing the genetically engineered bacteria.
6. Use of the mutant phosphoglucose kinase as claimed in claim 1, characterized in that: the method is applied to a phosphate transfer reaction, takes polyphosphoric acid as a phosphate donor, phosphorylates D-arabinose shown in a formula a, and is further used for preparing beta-D-arabinose nucleosides shown in a formula b, a formula c, a formula D, a formula e and a formula f;
CN202311176882.2A 2023-09-13 2023-09-13 Polyglucose kinase mutant and application thereof Pending CN117402851A (en)

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