In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(E...In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(EAC)and Bordetella Pertussis(BAC)were expressed in E.coli BL21(DE3)and comparatively analyzed for their activities.As a result,EAC from E.coli MG1655 exhibited a higher activity.However,amount of EAC were obtained in an insoluble form.Therefore,we expressed the first 446 amino acids of EAC(EAC446)to avoid the inclusion body.The effects of induction temperature,incubation time,and incubation p H were further evaluated to improve the expression of EAC446.Subsequently,the reaction process for the production of c AMP with ATP as a starting material was investigated.As none of c AMP was detected in the whole-cell based biocatalytic process,the reaction catalyzed by the crude enzyme was determined for c AMP production.What's more,the reaction temperature,reaction p H,metal ion additives and substrate concentration was optimized,and the maximum c AMP production of 18.45 g·L^-1was achieved with a yield of 95.4%after bioconversion of 6 h.展开更多
cAMP(环磷酸腺苷)是一种广泛存在于生物体内的小分子高生物活性物质和第二次信使,已有研究表明,外源c AMP对草本植物的种子萌发和生长发育具有重要影响,但木本植物中外源c AMP的应用效果鲜有报道。以酸枣仁和当年生酸枣实生苗为试材,用...cAMP(环磷酸腺苷)是一种广泛存在于生物体内的小分子高生物活性物质和第二次信使,已有研究表明,外源c AMP对草本植物的种子萌发和生长发育具有重要影响,但木本植物中外源c AMP的应用效果鲜有报道。以酸枣仁和当年生酸枣实生苗为试材,用不同浓度c AMP对酸枣仁进行浸种处理并对当年生酸枣实生苗进行根施、喷施c AMP处理,研究不同浓度外源c AMP对酸枣种子萌发和生长发育的影响。结果表明:25μmol/L c AMP浸种18 h,可极显著提高酸枣种子的萌发率(达90.69%);根施25μmol/L c AMP能够显著促进酸枣实生苗地上部生长;喷施50μmol/L c AMP对酸枣实生苗根系的生长有显著促进作用;喷施50μmol/L c AMP可极显著提高当年生酸枣实生苗开花率;根施25、50μmol/L c AMP均可显著提高当年生酸枣实生苗的结果率。该研究可为c AMP在果树上生产的应用及揭示其调控木本植物生长发育的分子机制提供借鉴和参考。展开更多
The unicellular green alga,Chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid metabolism.To find some novel genes regulating the...The unicellular green alga,Chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid metabolism.To find some novel genes regulating the lipid metabolism under various stress conditions,the paromomycin resistance gene aphVIII was transferred into the genome of C.reinhardtii to establish a mutant library.Two genes mutated in two of the TAG-reduced mutants(Cre06.g278111 in M2 mutant,Cre06.g278110 in M6 mutants)were neighboring in the genome,and their expression levels were down-regulated in their corresponding mutants in parallel with their reduced TAG levels following N deprivation.The proteins encoded by these two genes(KCN11 by Cre06.g278111,ACYC3 by Cre06.g278110)contained a conversed cyclic mononucleotide phosphate(cNMP)binding protein and an adenylate domain,respectively.Since cNMP binding protein and adenylate domain have been known as important components of cyclic adenosine monophosphate(cAMP)signaling pathway,suggesting that these two genes might af fect cellular TAG biosynthesis through cAMP signal pathway.展开更多
基金The National Natural Science Foundation of China(Grant No.21576134,Grant No.21606127,Grant No.21390200,Grant No.21706126)the National Key Research and Development Program of China(Grant No.2016YFA0204300)the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions。
文摘In this study,we aimed at developing an efficient biocatalytic process for bio-production of cyclic adenosine monophosphate(c AMP)from adenosine triphosphate(ATP).First,adenylate cyclase from Escherichia coli MG1655(EAC)and Bordetella Pertussis(BAC)were expressed in E.coli BL21(DE3)and comparatively analyzed for their activities.As a result,EAC from E.coli MG1655 exhibited a higher activity.However,amount of EAC were obtained in an insoluble form.Therefore,we expressed the first 446 amino acids of EAC(EAC446)to avoid the inclusion body.The effects of induction temperature,incubation time,and incubation p H were further evaluated to improve the expression of EAC446.Subsequently,the reaction process for the production of c AMP with ATP as a starting material was investigated.As none of c AMP was detected in the whole-cell based biocatalytic process,the reaction catalyzed by the crude enzyme was determined for c AMP production.What's more,the reaction temperature,reaction p H,metal ion additives and substrate concentration was optimized,and the maximum c AMP production of 18.45 g·L^-1was achieved with a yield of 95.4%after bioconversion of 6 h.
文摘cAMP(环磷酸腺苷)是一种广泛存在于生物体内的小分子高生物活性物质和第二次信使,已有研究表明,外源c AMP对草本植物的种子萌发和生长发育具有重要影响,但木本植物中外源c AMP的应用效果鲜有报道。以酸枣仁和当年生酸枣实生苗为试材,用不同浓度c AMP对酸枣仁进行浸种处理并对当年生酸枣实生苗进行根施、喷施c AMP处理,研究不同浓度外源c AMP对酸枣种子萌发和生长发育的影响。结果表明:25μmol/L c AMP浸种18 h,可极显著提高酸枣种子的萌发率(达90.69%);根施25μmol/L c AMP能够显著促进酸枣实生苗地上部生长;喷施50μmol/L c AMP对酸枣实生苗根系的生长有显著促进作用;喷施50μmol/L c AMP可极显著提高当年生酸枣实生苗开花率;根施25、50μmol/L c AMP均可显著提高当年生酸枣实生苗的结果率。该研究可为c AMP在果树上生产的应用及揭示其调控木本植物生长发育的分子机制提供借鉴和参考。
基金Supported by the National High Technology Research and Development Program of China(863 Program)(No.2014AA022001)
文摘The unicellular green alga,Chlamydomonas reinhardtii is a model organism for studying various biological processes,such as photosynthesis,flagellar motility,and lipid metabolism.To find some novel genes regulating the lipid metabolism under various stress conditions,the paromomycin resistance gene aphVIII was transferred into the genome of C.reinhardtii to establish a mutant library.Two genes mutated in two of the TAG-reduced mutants(Cre06.g278111 in M2 mutant,Cre06.g278110 in M6 mutants)were neighboring in the genome,and their expression levels were down-regulated in their corresponding mutants in parallel with their reduced TAG levels following N deprivation.The proteins encoded by these two genes(KCN11 by Cre06.g278111,ACYC3 by Cre06.g278110)contained a conversed cyclic mononucleotide phosphate(cNMP)binding protein and an adenylate domain,respectively.Since cNMP binding protein and adenylate domain have been known as important components of cyclic adenosine monophosphate(cAMP)signaling pathway,suggesting that these two genes might af fect cellular TAG biosynthesis through cAMP signal pathway.
