技术领域Technical field
本发明属于创新霉素衍生物合成技术领域，具体涉及一种用于合成创新霉素衍生物的酶组合物及其应用。The invention belongs to the technical field of synthesizing novelmycin derivatives, and specifically relates to an enzyme composition for synthesizing novelmycin derivatives and its application.
背景技术Background technique
创新霉素是上世纪七十年代由中国科学家在济南游动放线菌中发现的一个全新抗生素，具有特殊的吲哚骈二氢噻喃环母核结构，以及全新的硫原子引入机制。创新霉素作为色氨酸结构类似物，能够靶向抑制细菌的色氨酰tRNA合成酶，经实验验证对大肠杆菌和志贺氏菌有显著的抑制效果，但其对绵羊的色氨酰tRNA合成酶没有抑制作用，可以作为一种新型抗生素的候选分子。但由于创新霉素的抗菌谱较窄，影响了创新霉素在临床上的广泛使用。Chuangchuangxinmycin is a brand-new antibiotic discovered by Chinese scientists in Jinan's swimming actinomycetes in the 1970s. It has a special indole-thiopyran ring core structure and a new sulfur atom introduction mechanism. As a structural analog of tryptophan, Chuangchuangmycin can target and inhibit bacterial tryptophanyl-tRNA synthetase. It has been experimentally verified to have a significant inhibitory effect on Escherichia coli and Shigella. However, it has a significant inhibitory effect on tryptophanyl-tRNA synthesis in sheep. The enzyme has no inhibitory effect and could serve as a candidate molecule for a new class of antibiotics. However, the narrow antibacterial spectrum of Chuangchuangmycin has affected its widespread clinical use.
目前主要通过化学合成法对创新霉素进行修饰获得创新霉素衍生物。但化学合成方法步骤繁琐，反应条件复杂难以控制，并且在化学合成中不可避免地需要用到一些环境污染试剂如巯基乙酸乙酯等。At present, novelmycin derivatives are mainly obtained by modifying novelmycin through chemical synthesis. However, the steps of the chemical synthesis method are cumbersome, the reaction conditions are complex and difficult to control, and some environmentally polluting reagents such as ethyl thioglycolate are inevitably needed in the chemical synthesis.
发明内容Contents of the invention
本发明的目的是提供一种用于合成创新霉素衍生物的酶组合物及其应用，以及一种用色氨酸衍生物为底物，通过本发明的酶组合物来合成创新霉素衍生物的方法，为筛选具有良好抗菌活性的创新霉素衍生物提供了先导化合物资源。The object of the present invention is to provide an enzyme composition for synthesizing novelmycin derivatives and its application, as well as an enzyme composition for synthesizing novelmycin derivatives using tryptophan derivatives as substrates. This chemical method provides lead compound resources for screening novel mycin derivatives with good antibacterial activity.
本发明首先提供一种酶组合物，所述的酶组合物包含有Cxm3、Cxm4、Cxm5、Cxm6、Cxm7、CxmM、Fdx和FdR蛋白酶；The invention first provides an enzyme composition, which contains Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases;
所述的Cxm3蛋白酶的氨基酸序列为SEQ ID NO:1，Cxm4蛋白酶的氨基酸序列为SEQID NO:2，Cxm5蛋白酶的氨基酸序列为SEQ ID NO:3，Cxm6蛋白酶的氨基酸序列为SEQ IDNO:4，Cxm7蛋白酶的氨基酸序列为SEQ ID NO:5，CxmM蛋白酶的氨基酸序列为SEQ ID NO:6，FdR蛋白酶的氨基酸序列为SEQ ID NO:7，Fdx蛋白酶的氨基酸序列为SEQ ID NO:8，但还可以使用与序列为SEQ ID NO:1-8蛋白的同源酶/同工酶来催化。The amino acid sequence of the Cxm3 protease is SEQ ID NO: 1, the amino acid sequence of the Cxm4 protease is SEQ ID NO: 2, the amino acid sequence of the Cxm5 protease is SEQ ID NO: 3, the amino acid sequence of the Cxm6 protease is SEQ ID NO: 4, Cxm7 The amino acid sequence of the protease is SEQ ID NO:5, the amino acid sequence of the CxmM protease is SEQ ID NO:6, the amino acid sequence of the FdR protease is SEQ ID NO:7, the amino acid sequence of the Fdx protease is SEQ ID NO:8, but it can Catalysis is performed using homologous enzymes/isoenzymes with the protein sequence of SEQ ID NO: 1-8.
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；The enzyme composition provided by the invention is used to synthesize nornomycin derivatives using tryptophan derivatives as substrates through the multi-enzyme combination system of the invention;
所述的色氨酸衍生物，为5-氟色氨酸(1)、5-氯色氨酸(2)、5-溴色氨酸(3)、5-甲基色氨酸(4)、6-氟色氨酸(5)、6-氯色氨酸(6)、6-溴色氨酸(7)、6-甲基色氨酸(8)和7-甲氧色氨酸(9)。The tryptophan derivatives are 5-fluorotryptophan (1), 5-chlorotryptophan (2), 5-bromotryptophan (3), and 5-methyltryptophan (4) , 6-fluorotryptophan (5), 6-chlorotryptophan (6), 6-bromotryptophan (7), 6-methyltryptophan (8) and 7-methoxytryptophan ( 9).
所述的去甲创新霉素衍生物，为5-氟去甲创新霉素(a)、5-氯去甲创新霉素(b)、5-溴去甲创新霉素(c)、5-甲基去甲创新霉素(d)、6-氟去甲创新霉素(e)、6-氯去甲创新霉素(f)、6-溴去甲创新霉素(g)、6-甲基去甲创新霉素(h)和7-甲氧去甲创新霉素(i)。The described norcetomycin derivatives are 5-fluoronorcetomycin (a), 5-chloronocetomycin (b), 5-bromonocetomycin (c), 5- Methyl nornocetomycin (d), 6-fluoronorcetomycin (e), 6-chloronocetomycin (f), 6-bromonoremycin (g), 6-methyl nocetomycin 1-Methoxynorsinomycin (h) and 7-methoxynosinomycin (i).
作为实施例的记载，是从色氨酸衍生物1-9制备去甲创新霉素衍生物a-i。As an example, nornomycin derivatives a-i were prepared from tryptophan derivatives 1-9.
本发明的方法，是在反应缓冲液中添加色氨酸衍生物、上述的酶组合物、NADPH、ATP、Na2S2O3、MgCl2、丙酮酸钠、磷酸吡哆醛(PLP)和DTT来进行创新霉素衍生物的合成；The method of the present invention is to add tryptophan derivatives, the above-mentioned enzyme composition, NADPH, ATP, Na2 S2 O3 , MgCl2 , sodium pyruvate, pyridoxal phosphate (PLP) and DTT is used for the synthesis of novelmycin derivatives;
所述的反应缓冲液，其一种组成如下：NaH2PO4 50mM、NaCl 300mM，pH8.0。The reaction buffer has the following composition: NaH2 PO4 50mM, NaCl 300mM, pH 8.0.
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 solves the problem of using other tryptophan derivatives to synthesize nornomycin derivatives. Compared with chemical synthesis, enzymatic synthesis has strong specificity, mild and easy-to-control reaction conditions, simple operation, and is environmentally friendly. advantage.
附图说明Description of the drawings
图1：色氨酸衍生物的化学结构图，Figure 1: Chemical structure diagram of tryptophan derivatives,
图2：5-氟去甲创新霉素的结构和HPLC-MS检测图谱，Figure 2: Structure and HPLC-MS detection spectrum of 5-fluoronornomycin.
图3：5-氯去甲创新霉素的结构和HPLC-MS检测图谱，Figure 3: Structure and HPLC-MS detection spectrum of 5-chloronornomycin.
图4：5-溴去甲创新霉素的结构和HPLC-MS检测图谱，Figure 4: Structure and HPLC-MS detection spectrum of 5-bromonornomycin.
图5：5-甲基去甲创新霉素的结构和HPLC-MS检测图谱，Figure 5: Structure and HPLC-MS detection spectrum of 5-methylnornomycin.
图6：6-氟去甲创新霉素的结构和HPLC-MS检测图谱，Figure 6: The structure and HPLC-MS detection spectrum of 6-fluorodenomycin.
图7：6-氯去甲创新霉素的结构和HPLC-MS检测图谱，Figure 7: Structure and HPLC-MS detection spectrum of 6-chloronornomycin.
图8：6-溴去甲创新霉素的结构和HPLC-MS检测图谱，Figure 8: Structure and HPLC-MS detection spectrum of 6-bromonornomycin.
图9：6-甲基去甲创新霉素的结构和HPLC-MS检测图谱，Figure 9: Structure and HPLC-MS detection spectrum of 6-methylnornomycin.
图10：7-甲氧去甲创新霉素的结构和HPLC-MS检测图谱。Figure 10: The structure and HPLC-MS detection spectrum of 7-methoxynornomycin.
具体实施方式Detailed ways
本发明首先提供一种酶组合物，所述的酶组合物包含有Cxm3、Cxm4、Cxm5、Cxm6、Cxm7、CxmM、Fdx和FdR蛋白酶；The invention first provides an enzyme composition, which contains Cxm3, Cxm4, Cxm5, Cxm6, Cxm7, CxmM, Fdx and FdR proteases;
其中Cxm3的氨基酸序列为SEQ ID NO:1，Cxm4的氨基酸序列为SEQ ID NO:2，Cxm5的氨基酸序列为SEQ ID NO:3，Cxm6的氨基酸序列为SEQ ID NO:4，Cxm7的氨基酸序列为SEQID NO:5，CxmM的氨基酸序列为SEQ ID NO:6，FdR的氨基酸序列为SEQ ID NO:7，Fdx的氨基酸序列为SEQ ID NO:8。The amino acid sequence of Cxm3 is SEQ ID NO:1, the amino acid sequence of Cxm4 is SEQ ID NO:2, the amino acid sequence of Cxm5 is SEQ ID NO:3, the amino acid sequence of Cxm6 is SEQ ID NO:4, and the amino acid sequence of Cxm7 is SEQ ID NO:5, the amino acid sequence of CxmM is SEQ ID NO:6, the amino acid sequence of FdR is SEQ ID NO:7, and the amino acid sequence of Fdx is SEQ ID NO:8.
但还可使用与序列为SEQ ID NO:1-8的蛋白的同源酶/同工酶来进行催化。However, homologous enzymes/isoenzymes with the proteins of SEQ ID NO: 1-8 can also be used for catalysis.
本发明所提供的酶组合物能够以色氨酸衍生物为底物通过体外“一锅法”实现去甲创新霉素衍生物的合成。The enzyme composition provided by the invention can realize the synthesis of nornomycin derivatives through an in vitro "one-pot method" using tryptophan derivatives as substrates.
其中色氨酸衍生物(结构见图1)主要为吲哚环C5、C6位卤素原子(F、Cl、Br)和甲基取代以及C7氧甲基取代。Among them, tryptophan derivatives (the structure is shown in Figure 1) are mainly substituted by halogen atoms (F, Cl, Br) and methyl groups at the C5 and C6 positions of the indole ring and C7 oxygen methyl group.
所述的合成方法，是在反应缓冲液中添加色氨酸衍生物、上述的酶组合物、NADPH、ATP、Na2S2O3、MgCl2、丙酮酸钠、磷酸吡哆醛(PLP)和DTT来进行多种去甲创新霉素衍生物的合成；The synthesis method is to add tryptophan derivatives, the above-mentioned enzyme composition, NADPH, ATP, Na2 S2 O3 , MgCl2 , sodium pyruvate, and pyridoxal phosphate (PLP) to the reaction buffer. and DTT for the synthesis of a variety of nonomycin derivatives;
所述的反应缓冲液是可以保证酶反应活性和所添加的其他物质溶解性的盐溶液。其中一种具体的组分是NaH2PO4 50mM、NaCl 100-300mM，glycerol0-10％，pH 7.0-8.0。但还可以使用其它能发挥酶组合物功效的缓冲液。The reaction buffer is a salt solution that can ensure the enzyme reaction activity and the solubility of other added substances. One specific component is NaH2 PO4 50mM, NaCl 100-300mM, glycerol 0-10%, pH 7.0-8.0. However, other buffers capable of exerting the effect of the enzyme composition may also be used.
所述酶组合物及其它辅因子成分的浓度为1-10μM Cxm3、1-10μM Cxm4、1-10μMCxm5、1-10μM Cxm6、1-10μM Cxm7、1-10μM CxmM、5-50μM FdR、10-100μM Fdx、1-10mMNADPH、0.5-2mM ATP、0.5-2mM Na2S2O3、1-5mM MgCl2、1-3mM丙酮酸钠、1-20μM磷酸吡哆醛(PLP)和1-5mM DTT。The concentrations of the enzyme composition and other cofactor components are 1-10 μM Cxm3, 1-10 μM Cxm4, 1-10 μM Cxm5, 1-10 μM Cxm6, 1-10 μM Cxm7, 1-10 μM CxmM, 5-50 μM FdR, 10-100 μM Fdx, 1-10mMNADPH, 0.5-2mM ATP, 0.5-2mM Na2 S2 O3 , 1-5mM MgCl2 , 1-3mM sodium pyruvate, 1-20μM pyridoxal phosphate (PLP) and 1-5mM DTT.
下面结合实施例和附图对本发明进行详细的描述。The present invention will be described in detail below with reference to the embodiments and drawings.
实施例1：“一锅法”合成5-氟去甲创新霉素Example 1: "One-pot method" synthesis of 5-fluoronornomycin
(1)蛋白的诱导表达(1) Induced expression of protein
将高效表达Cxm3、Cxm4、Cxm5、Cxm6、CxmM、Fdx和FdR蛋白的大肠杆菌菌株划线接种至LB(含50μg/mL卡那霉素和34μg/mL氯霉素)平板，高效表达Cxm7蛋白的大肠杆菌接种至含50μg/mL卡那霉素的LB平板，37℃培养24h。挑取单克隆于含50μg/mL卡那霉素或含50μg/mL卡那霉素和34μg/mL氯霉素的50mL LB培养基，37℃、220rpm摇床培养过夜。按1％接种量接种至含相应抗性的500mL TB或LB培养基中，37℃、200rpm培养至OD600介于0.8～1，加入终浓度200μM的IPTG、500μM的5-氨基乙酰丙酸(5-ALA)和500μM的维生素B1(VB1)，随后，在16℃、150rpm条件下培养18-20h诱导蛋白表达。E. coli strains that highly express Cxm3, Cxm4, Cxm5, Cxm6, CxmM, Fdx and FdR proteins were streaked into LB plates (containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol). Escherichia coli was inoculated into LB plates containing 50 μg/mL kanamycin and cultured at 37°C for 24 h. Pick single clones and culture them in 50 mL LB medium containing 50 μg/mL kanamycin or 50 μg/mL kanamycin and 34 μg/mL chloramphenicol on a shaking table at 37°C and 220 rpm overnight. Inoculate 1% of the inoculum into 500 mL TB or LB medium containing the corresponding resistance, culture at 37°C and 200 rpm until the OD600 is between 0.8 and 1, add a final concentration of 200 μM IPTG and 500 μM 5-aminolevulinic acid (5 -ALA) and 500 μM vitamin B1 (VB1), and then cultured at 16°C and 150 rpm for 18-20 h to induce protein expression.
(2)蛋白的纯化(2)Protein purification
离心收集菌体，用Lysis buffer(50mM NaH2PO4，300mM NaCl，10mM咪唑，10％甘油，pH 8.0)涡旋振荡重悬菌体，超声破碎细胞后10000rpm，4℃离心60min，上清液与Ni-NTA4℃孵育60min。然后，使用Wash buffer(50mM NaH2PO4，300mM NaCl，20mM咪唑，10％甘油，pH8.0)对杂蛋白进行洗脱；用Elution buffer(50mM NaH2PO4，300mM NaCl，250mM咪唑，10％甘油，pH 8.0)对目标蛋白进行洗脱，根据目标蛋白大小选择合适的超滤管浓缩蛋白溶液；最后使用PD-10脱盐柱去除咪唑，缓冲溶液使用Desalting buffer(50mM NaH2PO4，300mMNaCl，10％甘油，pH 8.0)。Collect the cells by centrifugation, use Lysis buffer (50mM NaH2 PO4 , 300mM NaCl, 10mM imidazole, 10% glycerol, pH 8.0) to resuspend the cells by vortexing. After ultrasonic disruption of the cells, centrifuge at 10,000 rpm for 60 minutes at 4°C and remove the supernatant. Incubate with Ni-NTA at 4°C for 60 minutes. Then, use Wash buffer (50mM NaH2 PO4 , 300mM NaCl, 20mM imidazole, 10% glycerol, pH 8.0) to elute impurity proteins; use Elution buffer (50mM NaH2 PO4 , 300mM NaCl, 250mM imidazole, 10 % glycerol, pH 8.0) to elute the target protein, select an appropriate ultrafiltration tube to concentrate the protein solution according to the size of the target protein; finally use a PD-10 desalting column to remove imidazole, and use Desalting buffer (50mM NaH2 PO4 , 300mM NaCl) as the buffer solution. , 10% glycerol, pH 8.0).
(3)“一锅法”反应合成5-氟去甲创新霉素(3) "One-pot" reaction to synthesize 5-fluoronornomycin
以1mM 5-氟色氨酸(1)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为NaH2PO4 50mM、NaCl300mM，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。Using 1mM 5-fluorotryptophan (1) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is NaH2 PO4 50mM, NaCl 300mM, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction.
反应产物经HPLC-MS检测，结果显示化合物1经多酶催化后相应产生了与预期分子量相符的5-氟去甲创新霉素即吸收峰a准分子离子峰m/z238.0341[M+H]+(图2)，与氟代去甲创新霉素衍生物分子量238.0338[M+H]相符。The reaction product was detected by HPLC-MS, and the results showed that compound 1 was catalyzed by multiple enzymes and produced 5-fluorodenomycin with the expected molecular weight, that is, the absorption peak a quasi-molecular ion peak m/z238.0341 [M+H ]+ (Figure 2), which is consistent with the molecular weight of fluorodenomycin derivatives: 238.0338[M+H].
实施例2：“一锅法”合成5-氯去甲创新霉素Example 2: "One-pot method" synthesis of 5-chloronornomycin
以1mM 5-氯色氨酸(2)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物2经多酶催化后相应产生了与预期分子量相符的5-氯去甲创新霉素即吸收峰b准分子离子峰m/z254.0047[M+H]+(图3)，与氯代去甲创新霉素衍生物分子量254.0043[M+H]+一致。Using 1mM 5-chlorotryptophan (2) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 2 was catalyzed by multiple enzymes to produce 5-chloronornomycin with the expected molecular weight, that is, the absorption peak b quasi-molecular ion peak m/z 254.0047 [M+H]+ (Figure 3), which is consistent with the molecular weight of chloronornomycin derivative 254.0043[M+H]+ .
实施例3：“一锅法”合成5-溴去甲创新霉素Example 3: "One-pot method" synthesis of 5-bromonornomycin
以1mM 5-溴色氨酸(3)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物3经多酶催化后相应产生了与预期分子量相符的5-溴去甲创新霉素即吸收峰c准分子离子峰m/z297.9537[M+H]+(图4)，与溴代去甲创新霉素衍生物分子量297.9537[M+H]+一致。Using 1mM 5-bromotryptophan (3) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 3 was catalyzed by multiple enzymes to produce 5-bromononomycin with the expected molecular weight, that is, the absorption peak c quasi-molecular ion peak m/z 297.9537 [M+H]+ (Figure 4), which is consistent with the molecular weight of bromonoclomycin derivative 297.9537[M+H]+ .
实施例4：“一锅法”合成5-甲基去甲创新霉素Example 4: "One-pot method" synthesis of 5-methylnornomycin
以1mM 5-甲基色氨酸(4)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μMCxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物4经多酶催化后相应产生了与预期分子量相符的5-甲基去甲创新霉素即吸收峰d准分子离子峰m/z 234.0592[M+H]+(图5)，与甲基代去甲创新霉素衍生物分子量234.0589[M+H]+一致。Using 1mM 5-methyltryptophan (4) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 4 was catalyzed by multiple enzymes and produced 5-methylnornonomycin, which was consistent with the expected molecular weight, that is, the absorption peak d and the quasi-molecular ion peak m/z 234.0592 [M+H]+ (Figure 5), which is consistent with the molecular weight of methyl-nonomycin derivative 234.0589[M+H]+ .
实施例5：“一锅法”合成6-氟去甲创新霉素Example 5: "One-pot method" synthesis of 6-fluoronornomycin
以1mM 6-氟色氨酸(5)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为NaH2PO4 50mM、NaCl300mM，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，结果显示化合物5经多酶催化后相应产生了与预期分子量相符的5-氟去甲创新霉素即吸收峰e准分子离子峰m/z238.0333[M+H]+(图6)，与氟代去甲创新霉素衍生物分子量238.0338[M+H]+相符。Using 1mM 6-fluorotryptophan (5) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is NaH2 PO4 50mM, NaCl 300mM, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, and the results showed that compound 5 was catalyzed by multiple enzymes and produced 5-fluorodenomycin, which was consistent with the expected molecular weight, that is, the absorption peak e quasi-molecular ion peak m/z238.0333 [M+H ]+ (Figure 6), which is consistent with the molecular weight of fluorodenomycin derivative 238.0338[M+H]+ .
实施例6：“一锅法”合成6-氯去甲创新霉素Example 6: "One-pot method" synthesis of 6-chloronornomycin
以1mM 6-氯色氨酸(6)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为NaH2PO4 50mM、NaCl300mM，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，结果显示化合物6经多酶催化后相应产生了与预期分子量相符的6-氯去甲创新霉素即吸收峰f准分子离子峰m/z254.0040[M+H]+(图7)，与氯代去甲创新霉素衍生物分子量254.0043[M+H]+相符。Using 1mM 6-chlorotryptophan (6) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is NaH2 PO4 50mM, NaCl 300mM, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, and the results showed that compound 6 was catalyzed by multiple enzymes to produce 6-chloronornomycin with the expected molecular weight, that is, the absorption peak f quasi-molecular ion peak m/z254.0040 [M+H ]+ (Figure 7), which is consistent with the molecular weight of chloronornomycin derivative 254.0043[M+H]+ .
实施例7：“一锅法”合成6-溴去甲创新霉素Example 7: "One-pot method" synthesis of 6-bromonornomycin
以1mM 6-溴色氨酸(7)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μM Cxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物7经多酶催化后相应产生了与预期分子量相符的6-溴去甲创新霉素即吸收峰g准分子离子峰m/z 297.9540[M+H]+(图8)，与溴代去甲创新霉素衍生物分子量297.9537[M+H]+一致。Using 1mM 6-bromotryptophan (7) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 7 was catalyzed by multiple enzymes to produce 6-bromononomycin with the expected molecular weight, that is, the absorption peak g quasi-molecular ion peak m/z 297.9540 [M+H]+ (Figure 8), which is consistent with the molecular weight of bromonornomycin derivatives: 297.9537[M+H]+ .
实施例8：“一锅法”合成6-甲基去甲创新霉素Example 8: "One-pot method" synthesis of 6-methylnornomycin
以1mM 6-甲基色氨酸(8)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μMCxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物8经多酶催化后相应产生了与预期分子量相符的6-甲基去甲创新霉素即吸收峰h准分子离子峰m/z 234.0592[M+H]+(图9)，与甲基代去甲创新霉素衍生物分子量234.0589[M+H]+一致。Using 1mM 6-methyltryptophan (8) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 8 was catalyzed by multiple enzymes and produced 6-methylnornonomycin, which was consistent with the expected molecular weight, that is, the absorption peak h, the quasi-molecular ion peak m/z 234.0592 [M+H]+ (Figure 9), which is consistent with the molecular weight of methyl-nonomycin derivative 234.0589[M+H]+ .
实施例9：“一锅法”合成7-甲氧去甲创新霉素Example 9: "One-pot method" synthesis of 7-methoxynornomycin
以1mM 7-甲氧色氨酸(9)为底物，加入10μM Cxm3、10μM Cxm4、10μMCxm5、10μMCxm6、10μM Cxm7、10μM CxmM、50μM FdR、100μM Fdx、10mM NADPH、2mM ATP、2mM Na2S2O3、5mMMgCl2、3mM丙酮酸钠、20μM磷酸吡哆醛(PLP)和5mM DTT，反应缓冲液为50mM NaH2PO4、300mMNaCl，pH 8.0，总体积100μL，30℃反应6h后，向反应液中加入2倍体积甲醇终止反应。反应产物经HPLC-MS检测，显示化合物9经多酶催化后相应产生了与预期分子量相符的6-甲基去甲创新霉素即吸收峰i准分子离子峰m/z 250.0537[M+H]+(图10)，与甲基代去甲创新霉素衍生物分子量250.0538[M+H]+一致。Using 1mM 7-methoxytryptophan (9) as substrate, add 10μM Cxm3, 10μM Cxm4, 10μM Cxm5, 10μM Cxm6, 10μM Cxm7, 10μM CxmM, 50μM FdR, 100μM Fdx, 10mM NADPH, 2mM ATP, 2mM Na2 S2 O3 , 5mMgCl2 , 3mM sodium pyruvate, 20μM pyridoxal phosphate (PLP) and 5mM DTT, the reaction buffer is 50mM NaH2 PO4 , 300mM NaCl, pH 8.0, the total volume is 100μL, after reacting at 30°C for 6 hours, add Add 2 times the volume of methanol to the solution to terminate the reaction. The reaction product was detected by HPLC-MS, which showed that compound 9 was catalyzed by multiple enzymes and produced 6-methylnornonomycin with the expected molecular weight, that is, the absorption peak i quasi-molecular ion peak m/z 250.0537 [M+H]+ (Figure 10), which is consistent with the molecular weight of methyl-nonomycin derivative 250.0538[M+H]+ .
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661397.8ACN116836959B (en) | 2023-06-05 | 2023-06-05 | Enzyme composition for synthesizing neomycin derivative and application thereof |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310661397.8ACN116836959B (en) | 2023-06-05 | 2023-06-05 | Enzyme composition for synthesizing neomycin derivative and application thereof |
| Publication Number | Publication Date |
|---|---|
| CN116836959Atrue CN116836959A (en) | 2023-10-03 |
| CN116836959B CN116836959B (en) | 2025-07-29 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310661397.8AActiveCN116836959B (en) | 2023-06-05 | 2023-06-05 | Enzyme composition for synthesizing neomycin derivative and application thereof |
| Country | Link |
|---|---|
| CN (1) | CN116836959B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109266662A (en)* | 2017-07-18 | 2019-01-25 | 中国医学科学院医药生物技术研究所 | The gene cluster of one group of biosynthesis creatmycin or open loop creatmycin |
| WO2019173797A1 (en)* | 2018-03-08 | 2019-09-12 | New Atlas Biotechnologies Llc | Processes for the production of tryptamines |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN109266662A (en)* | 2017-07-18 | 2019-01-25 | 中国医学科学院医药生物技术研究所 | The gene cluster of one group of biosynthesis creatmycin or open loop creatmycin |
| WO2019173797A1 (en)* | 2018-03-08 | 2019-09-12 | New Atlas Biotechnologies Llc | Processes for the production of tryptamines |
| Title |
|---|
| XINGWANG ZHANG 等: ""Biosynthesis of Chuangxinmycin Featuring a Deubiquitinase-like Sulfurtransferase"", 《ANGEW CHEM INT ED ENGL》, vol. 60, no. 46, 7 October 2021 (2021-10-07), pages 24418 - 24423* |
| 侍媛媛 等: ""创新霉素生物合成研究进展"", 《中国抗生素杂志》, vol. 48, no. 3, 3 April 2023 (2023-04-03), pages 343 - 350* |
| Publication number | Publication date |
|---|---|
| CN116836959B (en) | 2025-07-29 |
| Publication | Publication Date | Title |
|---|---|---|
| Ravikumar et al. | Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications | |
| US20170152287A1 (en) | Methods and compositions for site-specific labeling of peptides and proteins | |
| Patel et al. | Periplasmic expression of carbonic anhydrase in Escherichia coli: a new biocatalyst for CO2 hydration | |
| CN110551771B (en) | Synthesis method of chiral 3-amino-1-butanol | |
| Hervé et al. | Biochemical characterization and physiological properties of Escherichia coli UDP-N-acetylmuramate: L-alanyl-γ-D-glutamyl-meso-diaminopimelate ligase | |
| CN105543191A (en) | Esterase PHE21 and encoding gene and application thereof | |
| CN112673100B (en) | Compositions comprising non-standard amino acids and uses thereof | |
| EP3330282A1 (en) | Cipa, cipb and pixa as scaffolds to organize proteins into crystalline inclusions | |
| WO2021050371A1 (en) | Biotin synthases for efficient production of biotin | |
| CN116836959A (en) | An enzyme composition for synthesizing novelmycin derivatives and its application | |
| US6905849B1 (en) | Process for preparing clavam derivatives by using polypeptides having β-lactam synthetase activity | |
| Plaks et al. | Lipoic acid ligase-promoted bioorthogonal protein modification and immobilization | |
| CN105802935A (en) | Esterase PHE14 as well as encoding gene and application thereof | |
| US10988742B2 (en) | Penicillin expandase mutants, DNA coding the mutants, reagent kit containing the mutants and the application | |
| WO2024160843A1 (en) | Mutant ketoreductase with increased ketoreductase activity as well as methods and uses involving the same | |
| TW201033367A (en) | Enzymatic production of cyclic di-guanosine monophosphate | |
| CN113234696B (en) | Method for synthesizing crocetin in vitro | |
| CN116769848B (en) | Biosynthesis method and application of selenium-containing compound | |
| JP2021003034A (en) | Coliform bacillus that expresses efp protein, and method for producing flavonoid compound by using the same | |
| CN114686467B (en) | Nanometer immobilization method based on protein trans-splicing, application and immobilized enzyme | |
| ES2272115B1 (en) | PRODUCTION OF CHEMICAL FORMS OF CEPHALOSPORINE C ACETYLASE. | |
| CN119120440A (en) | Glucose isomerase and method for improving glucose isomerase activity | |
| CN108410896A (en) | A kind of method that enzyme process prepares acetyl coenzyme A | |
| Li | Biosynthesis of Alterochromide Natural Products in Pseudoalteromonas piscicida JCM 20779 | |
| CN117535251A (en) | Enzymatic synthesis method of immunosuppressant mycophenolic acid and derivative thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |