CN116836959B - Enzyme composition for synthesizing neomycin derivative and application thereof - Google Patents
Enzyme composition for synthesizing neomycin derivative and application thereofInfo
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Abstract
本发明提供一种用于合成创新霉素衍生物的酶组合物及其应用，所述的酶组合物，所述的酶组合物包含有Cxm3、Cxm4、Cxm5、Cxm6、Cxm7、CxmM、Fdx和FdR蛋白酶。本发明所提供的酶组合物用于将色氨酸衍生物为底物，通过本发明的多酶组合体系来合成去甲创新霉素衍生物。本发明解决了利用其他色氨酸衍生物合成去甲创新霉素衍生物的方法，与化学合成相比，酶法合成具有专一性强，反应条件温和易控制，操作简单，对环境友好等优点。
The present invention provides an enzyme composition for synthesizing innovamycin derivatives and its use. The enzyme composition comprises Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx, and FdR proteases. The enzyme composition provided by the present invention is used to synthesize demethylinnovamycin derivatives using tryptophan derivatives as substrates via the multi-enzyme system of the present invention. The present invention provides a method for synthesizing demethylinnovamycin derivatives using other tryptophan derivatives. Compared with chemical synthesis, enzymatic synthesis has the advantages of high specificity, mild and easily controllable reaction conditions, simple operation, and environmental friendliness.
Description
Technical Field
The invention belongs to the technical field of synthesis of an innovative-mycin derivative, and particularly relates to an enzyme composition for synthesizing the novel-mycin derivative and application thereof.
Background
The novel antibiotic is a brand new antibiotic discovered in actinomycetes of the south China by the seventies of the last century, and has a special indolothiothiopyran ring parent nucleus structure and a novel sulfur atom introduction mechanism. The neomycin serving as a tryptophan structural analogue can target and inhibit bacterial tryptophanyl tRNA synthetase, has obvious inhibition effect on escherichia coli and shigella through experimental verification, has no inhibition effect on sheep tryptophanyl tRNA synthetase, and can be used as a candidate molecule of novel antibiotics. However, the narrow antibacterial spectrum of the neomycin affects the wide clinical application of the neomycin.
At present, the chemical synthesis method is mainly used for modifying the neomycin to obtain the neomycin derivative. However, the chemical synthesis method is complicated in steps, the reaction conditions are complicated and difficult to control, and some environmental pollution reagents such as ethyl thioglycolate and the like are inevitably required to be used in the chemical synthesis.
Disclosure of Invention
The invention aims to provide an enzyme composition for synthesizing an innovative derivative, application thereof and a method for synthesizing the innovative derivative by using tryptophan derivatives as substrates through the enzyme composition, which provide a lead compound resource for screening the innovative derivative with good antibacterial activity.
The present invention first provides an enzyme composition comprising Cxm, cxm, cxm, cxm, cxm7, cxmM, fdx and FdR protease;
The amino acid sequence of Cxm protease is SEQ ID NO. 1, the amino acid sequence of Cxm4 protease is SEQ ID NO. 2, the amino acid sequence of Cxm5 protease is SEQ ID NO. 3, the amino acid sequence of Cxm6 protease is SEQ ID NO. 4, the amino acid sequence of Cxm7 protease is SEQ ID NO. 5, the amino acid sequence of CxmM protease is SEQ ID NO. 6, the amino acid sequence of FdR protease is SEQ ID NO. 7, and the amino acid sequence of Fdx protease is SEQ ID NO. 8, but homologous enzymes/isoenzymes with the sequences of SEQ ID NO. 1-8 can also be used for catalysis.
The enzyme composition provided by the invention is used for synthesizing the nornovamycin derivative by taking the tryptophan derivative as a substrate and using the multienzyme combination system provided by the invention;
The tryptophan derivatives are 5-fluorotryptophan (1), 5-chlorotryptophan (2), 5-bromotryptophan (3), 5-methyltryptophan (4), 6-fluorotryptophan (5), 6-chlorotryptophan (6), 6-bromotryptophan (7), 6-methyltryptophan (8) and 7-methoxytryptophan (9).
The norneomycin derivatives are 5-fluoroneomycin (a), 5-chloronorneomycin (b), 5-bromonorneomycin (c), 5-methylnorneomycin (d), 6-fluoroneomycin (e), 6-chloronorneomycin (f), 6-bromonorneomycin (g), 6-methylnorneomycin (h) and 7-methoxynorneomycin (i).
As an example, the preparation of the desnovamycin derivatives a-i from tryptophan derivatives 1-9 is described.
The method of the present invention is to add tryptophan derivative, enzyme composition, NADPH, ATP, na 2S2O3、MgCl2, sodium pyruvate, pyridoxal phosphate (PLP) and DTT to the reaction buffer to synthesize the neomycin derivative;
The reaction buffer has a composition of NaH 2PO4 mM, naCl 300mM and pH8.0.
Compared with chemical synthesis, the method for synthesizing the norneomycin derivative by utilizing other tryptophan derivatives has the advantages of strong specificity, mild and easily controlled reaction conditions, simple operation, environmental friendliness and the like.
Drawings
FIG. 1 shows the chemical structure of tryptophan derivatives,
FIG. 2 Structure and HPLC-MS detection pattern of 5-fluoronorneomycin,
FIG. 3 Structure and HPLC-MS detection pattern of 5-chloronorneomycin,
FIG. 4 Structure and HPLC-MS detection pattern of 5-bromonorneomycin,
FIG. 5 Structure of 5-methyl-norneomycin and HPLC-MS detection pattern,
FIG. 6 Structure and HPLC-MS detection pattern of 6-fluoronorneomycin,
FIG. 7 Structure and HPLC-MS detection pattern of 6-chloronorneomycin,
FIG. 8 Structure and HPLC-MS detection pattern of 6-bromonorneomycin,
FIG. 9 Structure and HPLC-MS detection pattern of 6-methyl-norneomycin,
FIG. 10 Structure and HPLC-MS detection pattern of 7-methoxynorneomycin.
Detailed Description
The present invention first provides an enzyme composition comprising Cxm, cxm, cxm, cxm, cxm7, cxmM, fdx and FdR protease;
Wherein Cxm is SEQ ID NO 1, cxm4 is SEQ ID NO 2, cxm5 is SEQ ID NO 3, cxm6 is SEQ ID NO 4, cxm7 is SEQ ID NO 5, cxmM is SEQ ID NO 6, fdR is SEQ ID NO 7, fdx is SEQ ID NO 8.
But can also be catalyzed by homologous/isozymes to the proteins having the sequences SEQ ID NOS: 1-8.
The enzyme composition provided by the invention can realize the synthesis of the norneomycin derivative by taking the tryptophan derivative as a substrate through an in-vitro one-pot method.
Wherein tryptophan derivatives (structure shown in figure 1) are mainly indole ring C5, C6 halogen atoms (F, cl, br) and methyl substitution and C7 oxymethyl substitution.
The synthesis method comprises the steps of adding tryptophan derivatives, the enzyme composition, NADPH, ATP, na 2S2O3、MgCl2, sodium pyruvate, pyridoxal phosphate (PLP) and DTT into a reaction buffer solution to synthesize a plurality of nornovamycin derivatives;
The reaction buffer is a salt solution which can ensure the enzyme reaction activity and the solubility of other added substances. One specific component is NaH 2PO4 mM, naCl 100-300mM, glycol 0-10%, pH 7.0-8.0. However, other buffers that can exert the effects of the enzyme composition may also be used.
The enzyme composition and other cofactor components have a concentration of 1-10μM Cxm3、1-10μM Cxm4、1-10μM Cxm5、1-10μM Cxm6、1-10μM Cxm7、1-10μM CxmM、5-50μM FdR、10-100μM Fdx、1-10mM NADPH、0.5-2mM ATP、0.5-2mM Na2S2O3、1-5mM MgCl2、1-3mM sodium pyruvate, 1-20. Mu.M pyridoxal phosphate (PLP) and 1-5mM DTT.
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
EXAMPLE 1 Synthesis of 5-fluoronorneomycin by one pot method
(1) Inducible expression of proteins
Coli strains expressing Cxm, cxm, cxm, cxm, cxmM, fdx and FdR proteins were streaked onto LB (containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol) plates, and the coli strain expressing Cxm7 protein was streaked onto LB plates containing 50 μg/mL kanamycin and cultured at 37 ℃ for 24h. A single clone was picked up in 50mL LB medium containing 50. Mu.g/mL kanamycin or 50mL of kanamycin and 34. Mu.g/mL chloramphenicol, and shake cultured overnight at 37℃and 220 rpm. Inoculating to 500mL TB or LB medium containing corresponding resistance according to 1% inoculum size, culturing at 37 ℃ and 200rpm until OD600 is between 0.8 and 1, adding 200 mu M of IPTG, 500 mu M of 5-aminolevulinic acid (5-ALA) and 500 mu M of vitamin B1 (VB 1) to the final concentration, and then culturing at 16 ℃ and 150rpm for 18-20h to induce protein expression.
(2) Purification of proteins
The cells were collected by centrifugation, resuspended by vortexing with a Lysis buffer (50 mM NaH 2PO4, 300mM NaCl,10mM imidazole, 10% glycerol, pH 8.0), sonicated, centrifuged at 10000rpm at 4℃for 60min, and the supernatant incubated with Ni-NTA at 4℃for 60min. The protein mixture was then eluted using Wash buffer (50 mM NaH 2PO4, 300mM NaCl,20mM imidazole, 10% glycerol, pH 8.0), the target protein was eluted using an Elutation buffer (50 mM NaH 2PO4, 300mM NaCl,250mM imidazole, 10% glycerol, pH 8.0), the protein solution was concentrated using an ultrafiltration tube appropriate for the size of the target protein, and finally imidazole was removed using a PD-10 desalting column, and the buffer solution was DESALTING BUFFER (50 mM NaH 2PO4, 300mM NaCl,10% glycerol, pH 8.0).
(3) One-pot reaction to synthesize 5-fluoro-norneomycin
Using 1mM 5-fluorotryptophan (1) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 100. Mu.L with a reaction buffer of NaH 2PO4 mM, naCl300mM and pH 8.0, 2 volumes of methanol were added to the reaction solution to terminate the reaction.
The reaction product was detected by HPLC-MS, and as a result, compound 1 was catalyzed by multiple enzymes to produce 5-fluoronorneomycin corresponding to the expected molecular weight, namely, absorption peak a excimer ion peak M/z238.0341[ M+H ] + (FIG. 2), which corresponds to the fluoronorneomycin derivative molecular weight 238.0338[ M+H ].
EXAMPLE 2 Synthesis of 5-Chlorodeoxycycline by one pot method
Using 1mM 5-chlorotryptophan (2) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0 in a reaction buffer, 2 volumes of methanol were added to the reaction mixture to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 2, after multienzyme catalysis, correspondingly produced 5-chloronorneomycin, the absorption peak b, excimer ion peak M/z254.0047[ M+H ] + (FIG. 3), consistent with the expected molecular weight of the chloronorneomycin derivative, 254.0043[ M+H ] +.
EXAMPLE 3 Synthesis of 5-Bromonorneomycin by "one pot" method
Using 1mM 5-bromotryptophan (3) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0 in a reaction buffer, 2 volumes of methanol were added to the reaction mixture to terminate the reaction. The reaction product was tested by HPLC-MS and showed that compound 3, after multienzyme catalysis, correspondingly produced 5-bromonorneomycin, the absorption peak c, excimer ion peak M/z297.9537[ M+H ] + (FIG. 4), consistent with the expected molecular weight of the bromonorneomycin derivative, 297.9537[ M+H ] +.
EXAMPLE 4 one pot Synthesis of 5-methyl Demethyl novacemycin
Using 1mM 5-methyltryptophan (4) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0 in a reaction buffer, 2 volumes of methanol were added to the reaction mixture to terminate the reaction. The reaction product was tested by HPLC-MS and showed that compound 4, after multienzyme catalysis, correspondingly produced 5-methyl-nornovamycin, the absorption peak d excimer ion peak m/z 234.0592[ M+H ] + (FIG. 5), consistent with the expected molecular weight of the methyl-nornovamycin derivative, 234.0589[ M+H ] +.
EXAMPLE 5 Synthesis of 6-fluoro-norneomycin by one pot method
Using 1mM 6-fluorotryptophan (5) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 100. Mu.L with a reaction buffer of NaH 2PO4 mM, naCl300mM and pH 8.0, 2 volumes of methanol were added to the reaction solution to terminate the reaction. The reaction product was detected by HPLC-MS, and as a result, compound 5 was catalyzed by multiple enzymes to produce 5-fluoronorneomycin corresponding to the expected molecular weight, namely, absorption peak e excimer ion peak M/z238.0333[ M+H ] + (FIG. 6), which was consistent with the molecular weight of the fluoronorneomycin derivative 238.0338[ M+H ] +.
EXAMPLE 6 Synthesis of 6-Chlorodeoxycycline by one pot method
Using 1mM 6-chlorotryptophan (6) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 100. Mu.L with NaH 2PO4 mM, naCl300mM and pH 8.0, 2 volumes of methanol were added to the reaction solution to terminate the reaction. The reaction product was tested by HPLC-MS, and as a result, compound 6 was catalyzed by multiple enzymes to produce 6-chloronorneomycin corresponding to the expected molecular weight, namely, absorption peak f excimer ion peak M/z254.0040[ M+H ] + (FIG. 7), which was consistent with the molecular weight 254.0043[ M+H ] + of the chloronorneomycin derivative.
EXAMPLE 7 Synthesis of 6-Bromonorneomycin by "one pot" method
Using 1mM 6-bromotryptophan (7) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0 in a reaction buffer, 2 volumes of methanol were added to the reaction solution to terminate the reaction. The reaction product was tested by HPLC-MS and showed that compound 7, after multienzyme catalysis, correspondingly produced 6-bromonorneomycin, an absorption peak g excimer ion peak m/z 297.9540[ M+H ] + (FIG. 8), consistent with the expected molecular weight of the bromonorneomycin derivative, 297.9537[ M+H ] +.
EXAMPLE 8 Synthesis of 6-methyl Demethyl-novacells by one pot method
Using 1mM 6-methyltryptophan (8) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0 in a reaction buffer, 2 volumes of methanol were added to the reaction mixture to terminate the reaction. The reaction product was tested by HPLC-MS and showed that compound 8, after multienzyme catalysis, correspondingly produced 6-methyl-nornovamycin, the absorption peak h, excimer ion peak m/z 234.0592[ M+H ] + (FIG. 9), consistent with the molecular weight 234.0589[ M+H ] + of the methyl-nornovamycin derivative.
EXAMPLE 9 one pot Synthesis of 7-methoxynorneomycin
Using 1mM 7-methoxytryptophan (9) as a substrate, 10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mM MgCl2、3mM sodium pyruvate, 20. Mu.M pyridoxal phosphate (PLP) and 5mM DTT were added, and after a reaction of 6 hours at 30℃in a total volume of 50mM NaH 2PO4 and 300mM NaCl,pH 8.0, 2 volumes of methanol were added to the reaction mixture to terminate the reaction. The reaction product was examined by HPLC-MS and showed that compound 9, after multienzyme catalysis, correspondingly produced 6-methyl-nornovamycin, the absorption peak i, excimer ion peak m/z 250.0537[ M+H ] + (FIG. 10), consistent with the expected molecular weight, 250.0538[ M+H ] +, of the methyl-nornovamycin derivative.
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| CN107201356A (en) * | 2016-03-17 | 2017-09-26 | 中国科学院青岛生物能源与过程研究所 | Support the reduction chaperone combination of P450 decarboxylation of fatty acids enzymatic activitys and its apply |
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| CN106754986A (en) * | 2017-03-13 | 2017-05-31 | 山东大学苏州研究院 | Creatmycin biological synthesis gene cluster and its application |
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