自诱导系统在酶促合成2’-脱氧胞苷中的应用
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摘要
旨在高效合成2’-脱氧胞苷(d Cyd)。DCyd作为一类重要的药物中间体,能够用于合成吉西他滨(d Fd C)、扎西他滨(dd C)等多种抗病毒或抗肿瘤的药物。采用ZYM-Fe-5052自诱导培养基对含有N-脱氧核糖转移酶II(NDT)的大肠杆菌BL21(DE3)进行诱导,所得完整菌体直接用于催化合成d Cyd。结果表明,自诱导系统不仅无需额外添加诱导剂,还能够达到较高的菌体密度,且与LB诱导系统所得菌体在合成d Cyd方面具有同样高效的催化活性。2’-脱氧胸苷(d Thd)和胞嘧啶的浓度比为1∶3时,产物转化率可高达86.5%。合成d Cyd的最适反应条件为:2’-脱氧胸苷(d Thd)和胞嘧啶的浓度比为1∶1.5,p H6.5的磷酸缓冲液,1 mg/m L的菌体量,终体系10 m L,30℃条件下反应1 h,产物转化率可达71.9%。此方法还可用于制备其他核苷类似物,具有广泛的适用性和应用前景。
This experiment was carried out in order to synthesize 2'-deoxycytidine(d Cyd)more efficiently. DCyd is a significant intermediate for many antiviral and anticancer drugs such as Gemcitabine(d Fd C), Dideoxycytidine(dd C), etc. A convenient and efficient bioprocess was reported here to synthesize d Cyd by E.coli BL21(DE3)containing gene of N-deoxyribosyltransferase Ⅱ(NDT)overexpressed using ZYM-Fe-5052 auto-induction system. The results showed that auto-induction system could make the addition of inducer during cultivation unnecessary and obtain a higher cell density. The recombinant cells from auto-induction system were as efficient as that from LB system on the conversion yields of d Cyd. When the ratio of 2'-deoxythymidine(d Thd)and cytosine was 1∶3, the conversion yield could be 86.5%. The substrates ratio of 2'-deoxythymidine(d Thd) and cytosine as 1∶1.5 and 1 mg/m L recombinant cells which were dissolved in phosphate buffer of p H6.5, then cultured at 30℃ for 1 h were selected as the suitable reaction conditions,and the conversion yield could be 71.9%. This method also has potential for the preparation of other nucleoside analogues.
This experiment was carried out in order to synthesize 2'-deoxycytidine(d Cyd)more efficiently. DCyd is a significant intermediate for many antiviral and anticancer drugs such as Gemcitabine(d Fd C), Dideoxycytidine(dd C), etc. A convenient and efficient bioprocess was reported here to synthesize d Cyd by E.coli BL21(DE3)containing gene of N-deoxyribosyltransferase Ⅱ(NDT)overexpressed using ZYM-Fe-5052 auto-induction system. The results showed that auto-induction system could make the addition of inducer during cultivation unnecessary and obtain a higher cell density. The recombinant cells from auto-induction system were as efficient as that from LB system on the conversion yields of d Cyd. When the ratio of 2'-deoxythymidine(d Thd)and cytosine was 1∶3, the conversion yield could be 86.5%. The substrates ratio of 2'-deoxythymidine(d Thd) and cytosine as 1∶1.5 and 1 mg/m L recombinant cells which were dissolved in phosphate buffer of p H6.5, then cultured at 30℃ for 1 h were selected as the suitable reaction conditions,and the conversion yield could be 71.9%. This method also has potential for the preparation of other nucleoside analogues.
引文
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[17]Anand R,Kaminski PA,Ealick SE.Structures of purine 2￠-Deoxyribosyltransferase,substrate complexes,and the ribosylated enzyme intermediate at 2.0?resolution[J].Biochemistry,2004,43:2384-2393.
[18]Kiyoshi O,Toshitada N.Molecular cloning and expression of the nucleoside deoxyribosyltransferase-II Gene from lactobacillus helveticus[J].Biosci Biotechnol Biochem,2000,64:2243-2245.
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[20]Donovan RS,Robinson CW,Glick BR.Review:Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J].J Ind Microbiol,1996,16:145-154.
[21]Neubauer P,Hofmann K,Holst O,et al.Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer[J].Appl Microbiol Biotechnol,1992,36:739-744.
[22]Studier FW.Protein production by auto-induction in high-density shaking cultures[J].Protein Expres Purif,2005,41:207-234.
[23]Xiong J,Zhang W,Su J,et al.Improved synthesis of 2’-deoxyadenosine and 5-methyluridine by Escherichia coli using an auto-induction system[J].World J Microbiol Biotechnol,2012,28:721-727.
[24]Lin Y,Zhang W,Zhu F,et al.Subcellular localization of N-deoxyribosyltransferase in Lactobacillus fermentum:cell surface association of an intracellular nucleotide metabolic enzyme[J].FEMS Microbiol Lett,2011,323:132-141.
[25]Fernandez-Lucas J,Acebal C,Sinisterra JV,et al.Lactobacillus reuteri 2’-deoxyribosyltransferase,a novel biocatalyst for tailoring of nucleosides[J].Appl Environ Microb,2010,76:1462-1470.
[26]Britos CN,Cappa VA,Rivero CW,et al.Biotransformation of halogenated 2’-deoxyribosides by immobilized lactic acid bacteria[J].J Mol Catal B-Enzym,2012,79:49-53.
[2]严琳,孔军,侯莉莉.核苷类似物的设计合成及其抗肿瘤活性[J].河南大学学报:自然科学版,2011,41(3):257-261.
[3]Galmarini CM,Mackey JR,Dumontet C.Nucleoside analogues and nucleobases in cancer treatment[J].Lancet Oncol,2002,3:415-424.
[4]Mathe C,Gosselin G.L-nucleoside enantiomers as antivirals drugs:a mini-review[J].Antivir Res,2006,71:276-281.
[5]Zhang J,Visser F,King KM,et al.The role of nucleoside transporters in cancer chemotherapy with nucleoside drugs[J].Cancer Metast Rev,2007,26:85-110.
[6]Zou Z,Ding Q,Ou L,Yan B.Efficient production of deoxynucleoside-5’-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system[J].Appl Microbiol Biotechnol,2013,97:9389-9395.
[7]徐渊,王鸿,易喻,等.核苷合成技术的研究进展[J].化工生产与技术,2009,16(2):32-39.
[8]王丽慧,丁庆豹,欧伶,等.酶法合成5-杂氮-2’-脱氧胞苷[J].华东理工大学学报:自然科学报,2011,37(7):325-329.
[9]樊华伟,傅绍军,邵志宇,朱利民.核苷类药物酶法合成研究进展[J].生物技术通讯,2005,16(6):690-692.
[10]邱蔚然,丁庆豹.酶法合成核苷类抗病毒药物[J].中国医药工业杂志,1999,30(10):474-478.
[11]Macnutt WS.The enzymically catalysed transfer of the deoxyribosyl group from one purine or pyrimidine to another[J].Biochem J,1951,50:384-397.
[12]Holguin J,Cardinaud R.Trans-N-Deoxyribosylase:Purification by affinity chromatography and characterization[J].Eur J Biochem,1975,54:505-514.
[13]Holguin J,Cardinaud R.Trans-N-Deoxyribosylase:Substrate specificity studies purine bases as acceptors[J].Eur J Biochem,1975,54:515-520.
[14]Kaminski PA.Functional cloning,heterologous expression,and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus[J].J Biol Chem,2002,277:14400-14407.
[15]Carson DA,Wasson DB.Synthesis of 2’,3’-dideoxynucleosides by enzymatic trans-glycosylation[J].Biochem Bioph Res Co,1988,155:829-834.
[16]Freeman GA,Shaver SR,Rideout JL,Short SA.2-amino-9-(3-azido-2,3-dideoxy-β-D-erythro-pentofuranosyl)-6-substituted-9H-purines:Synthesis and anti-HIV activity[J].Bioorgan Med Chem,1995,3:447-458.
[17]Anand R,Kaminski PA,Ealick SE.Structures of purine 2￠-Deoxyribosyltransferase,substrate complexes,and the ribosylated enzyme intermediate at 2.0?resolution[J].Biochemistry,2004,43:2384-2393.
[18]Kiyoshi O,Toshitada N.Molecular cloning and expression of the nucleoside deoxyribosyltransferase-II Gene from lactobacillus helveticus[J].Biosci Biotechnol Biochem,2000,64:2243-2245.
[19]Gombert AK,Kilikian BV.Recombinant gene expression in Escherichia colicultivation using lactose as inducer[J].J Biotechnol,1998,60:47-54.
[20]Donovan RS,Robinson CW,Glick BR.Review:Optimizing inducer and culture conditions for expression of foreign proteins under the control of the lac promoter[J].J Ind Microbiol,1996,16:145-154.
[21]Neubauer P,Hofmann K,Holst O,et al.Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer[J].Appl Microbiol Biotechnol,1992,36:739-744.
[22]Studier FW.Protein production by auto-induction in high-density shaking cultures[J].Protein Expres Purif,2005,41:207-234.
[23]Xiong J,Zhang W,Su J,et al.Improved synthesis of 2’-deoxyadenosine and 5-methyluridine by Escherichia coli using an auto-induction system[J].World J Microbiol Biotechnol,2012,28:721-727.
[24]Lin Y,Zhang W,Zhu F,et al.Subcellular localization of N-deoxyribosyltransferase in Lactobacillus fermentum:cell surface association of an intracellular nucleotide metabolic enzyme[J].FEMS Microbiol Lett,2011,323:132-141.
[25]Fernandez-Lucas J,Acebal C,Sinisterra JV,et al.Lactobacillus reuteri 2’-deoxyribosyltransferase,a novel biocatalyst for tailoring of nucleosides[J].Appl Environ Microb,2010,76:1462-1470.
[26]Britos CN,Cappa VA,Rivero CW,et al.Biotransformation of halogenated 2’-deoxyribosides by immobilized lactic acid bacteria[J].J Mol Catal B-Enzym,2012,79:49-53.