拟南芥肌醇半乳糖苷合成酶与棉子糖合成酶的体外催化活性比较
|
摘要
[目的]基因克隆及原核表达纯化后比较拟南芥的2个肌醇半乳糖苷合成酶及2个棉子糖合成酶的体外催化活性,为微生物法或酶法合成棉子糖尊定基础。[方法]RT-PCR克隆拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)的基因,分别构建原核表达菌株,诱导表达纯化获得酶,电泳检测及蛋白定量后进行体外酶催化反应,HPLC分析产物。[结果]克隆到GolS1与GolS3及RafS1与RafS5的基因,原核表纯化获得纯酶,以反应体系中目标产物生成速率衡量,GolS1与GolS3催化速率分别为0.51和0.28mmol/(mg·min),RafS1与RafS5的催化速率分别为0.45和0.21mmol/(mg·min)。[结论]拟南芥的肌醇半乳糖苷合成酶(GolS1及GolS3)与棉子糖合成酶(RafS1及RafS5)基因经异源表达后具有良好酶活,其中GolS1酶活是GolS3的1.82倍,RafS1酶活是RafS5的2.14倍。
[Objective]Gene clone,prokaryotic expression and comparison of the catalytic activity in vitro for 2 galactinol synthases and 2 raffinose synthetases from Arabidopsis thaliana,as a fundamental research for the enzymatic synthesis or microbial synthesis of raffinose.[Method] RT-PCR was implemented to get the genes of the galactinol synthases( Gol S1 and Gol S3)and the raffinose synthetases( Raf S1 and Raf S5) from Arabidopsis thaliana. Those genes' recombinant strains were constructed. After inducible expression,the galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5)were purified. Enzyme purity and quantity were carried out with the SDS-PAGE electrophoresis and the protein quantitation kit. The enzyme catalyzed reactions were implemented in vitro and the products were detected by HPLC.[Result]Genes of the galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5) were successfully cloned. After prokaryotic expression and purification,enzyme purity and quantity meet the comparison of the catalytic activity in vitro. Measured by the rate of production of the target product in the reaction system,the catalytic rates of Gol S1 and Gol S3 were 0. 51 and 0. 28 mmol/( mg·min) respectively,as the same time Raf S1 and Raf S5 were 0. 45 and 0. 21 mmol/( mg·min) respectively.[Conclusion] The galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5) from Arabidopsis thaliana have good enzyme activity after heterologous expression and purification. Gol S1 activity is higher than Gol S3 as well as Raf S1 activity is higher than Raf S5,which activity of Gol S1 is 1. 82 times that of Gol S3,and which of Raf S1 is 2. 14 times that of Raf S5.
[Objective]Gene clone,prokaryotic expression and comparison of the catalytic activity in vitro for 2 galactinol synthases and 2 raffinose synthetases from Arabidopsis thaliana,as a fundamental research for the enzymatic synthesis or microbial synthesis of raffinose.[Method] RT-PCR was implemented to get the genes of the galactinol synthases( Gol S1 and Gol S3)and the raffinose synthetases( Raf S1 and Raf S5) from Arabidopsis thaliana. Those genes' recombinant strains were constructed. After inducible expression,the galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5)were purified. Enzyme purity and quantity were carried out with the SDS-PAGE electrophoresis and the protein quantitation kit. The enzyme catalyzed reactions were implemented in vitro and the products were detected by HPLC.[Result]Genes of the galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5) were successfully cloned. After prokaryotic expression and purification,enzyme purity and quantity meet the comparison of the catalytic activity in vitro. Measured by the rate of production of the target product in the reaction system,the catalytic rates of Gol S1 and Gol S3 were 0. 51 and 0. 28 mmol/( mg·min) respectively,as the same time Raf S1 and Raf S5 were 0. 45 and 0. 21 mmol/( mg·min) respectively.[Conclusion] The galactinol synthases( Gol S1 and Gol S3) and the raffinose synthetases( Raf S1 and Raf S5) from Arabidopsis thaliana have good enzyme activity after heterologous expression and purification. Gol S1 activity is higher than Gol S3 as well as Raf S1 activity is higher than Raf S5,which activity of Gol S1 is 1. 82 times that of Gol S3,and which of Raf S1 is 2. 14 times that of Raf S5.
引文
[1]Sengupta S,Mukherjee S,Basak P,et al.Significance of galactinol and raffinose family oligosaccharide synthesis in plants[J].Frontiers in Plant Science,2015,6:656.DOI.org/10.3389/fpls.2015.00656.
[2]Van den Ende W.Multifunctional fructans and raffinose family oligosaccharides[J].Frontiers in Plant Science,2013,4(4):247.DOI.org/10.3389/fpls.2013.00247.
[3]周英彪,田朝玉,朱玥明等.在酿酒酵母中重构棉子糖生物合成途径的基础研究[J].现代食品科技,2017,33(5):121-128.
[4]Adamberg K,Adamberg S,Ernits K,et al.Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose,raffinose and raffinose-derived fructans[J].Anaerobe,2018,52(8):100-110.
[5]朱庆莉,兰宏兵,云志.双液相溶剂浸出棉籽中棉子糖和棉酚的工艺[J].南京工业大学学报:自然科学版,2014,36(4):114-117.
[6]李芳,汪晓峰.植物中棉子糖系列寡糖代谢及其调控关键酶研究进展[J].西北植物学报,2008,28(4):852-859.
[7]Taji T,Ohsumi C,Iuchi S,et al.Important roles of drought-and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[J].The Plant Journal,2002,29(4):417-426.
[8]Egert A,Keller F,Peters S.Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase(RS5,At5g40390)[J].BMC Plant Biology,2013,13(1):218.DOI.org/10.1186/1471-2229-13-218.
[9]Gangola M P,Jaiswal S,Kannan U,et al.Galactinol synthase enzyme activity influences raffinose family oligosaccharides(RFO)accumulation in developing chickpea(Cicer arietinum L.)seeds[J].Phytochemistry,2016,125:88-98.
[10]Kannan U,Sharma R,Khedikar Y,et al.Differential expression of two galactinol synthase isoforms Lc GolS1 and Lc GolS2 in developing lentil(Lens culinaris Medik.cv CDC Redberry)seeds[J].Plant Physiology and Biochemistry,2016,108:422-433.
[11]谷雷.玉米肌醇半乳糖苷合成酶2基因(Zm GOLS2)的功能和表达调控研究[D].咸阳:西北农林科技大学,2018.
[12]Maruyama K,Urano K,Yoshiwara K,et al.Integrated analysis of the effects of cold and dehydration on rice metabolites,phytohormones,and gene transcripts[J].Plant Physiology,2014,164(4):1759-1771.
[13]乔梦,田原,杨瑞,等.小麦棉子糖合成酶基因(TaRS)的克隆及特性分析[J].农业生物技术学报,2017,25(8):1245-1254.
[14]左静.梅花肌醇半乳糖苷和棉子糖合成酶基因的克隆与功能初探[D].武汉:华中农业大学,2017.
[15]眭晓蕾.黄瓜棉子糖合成酶基因克隆、功能验证及其低温响应研究[C].中国园艺学会2011年学术年会论文集:中国园艺学会,2011:1.
[16]张瑞腾,吕建春,周梦迪等.甜瓜肌醇半乳糖苷合成酶基因CmGAS1的表达与功能分析[J].园艺学报,2018,45(10):1929-1940.
[17]冯睿杰,侯丽霞,郭扬等.葡萄肌醇半乳糖苷合成酶基因Vv GolS2-4的克隆及表达特性分析[J].华北农学报,2017,32(02):87-95.
[18]王毅,肖良俊,马婷等.泡核桃肌醇半乳糖苷合成酶基因克隆及表达分析[J].基因组学与应用生物学,2018,37(05):2029-2033.
[19]李娜.辣椒肌醇半乳糖苷合成酶基因克隆与低温表达分析[D].扬州:扬州大学,2008.
[20]王君珂.基于RNA-Seq分析甜菜碱和棉子糖代谢途径在小桐子干旱响应与适应过程中的作用[D].昆明:云南师范大学,2017.
[21]George S,Manoharan D,Li J,et al.Drought and salt stress in Macrotyloma uniflorum leads to common and specific transcriptomic responses and reveals importance of raffinose family oligosaccharides in stress tolerance[J].Gene Reports,2018,10:7-16.
[22]Jing Y,Lang S,Wang D,et al.Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds[J].Journal of Plant Physiology,2018,230:109-121.
[23]Tian C,Yang J,Zeng Y,et al.Biosynthesis of raffinose and stachyose from sucrose via an in vitro multienzyme system[J].Appl.Environ.Microbiol.,2018:AEM.02306-18.DOI:10.1128/AEM.02306-18.
[24]Pharr D M,Sox H N,Locy R D,et al.Partial characterization of the galactinol forming enzyme from leaves of Cucumis sativus L[J].Plant Science Letters,1981,23(1):25-33.
[25]Handlet L W,Pharr D M.Ion stimulation,UDP inhibition and effects of sulfhydryl reagents on the activity of galactinol synthase from leaves of cucumber,Cucumis sativus L[J].Zeitschrift für Pflanzenphysiologie,1982,108(5):447-455.
[26]Smith P T,Kuo T M,Crawford C G.Purification and characterization of galactinol synthase from mature zucchini squash leaves[J].Plant Physiology,1991,96(3):693-698.
[27]Hart D O,He S,Chany C J,et al.Identification of Asp-130 as the catalytic nucleophile in the mainα-galactosidase from Phanerochaete chrysosporium,a family 27 glycosyl hydrolase[J].Biochemistry,2000,39(32):9826-9836.
[28]Ly H D,Howard S,Shum K,et al.The synthesis,testing and use of 5-fluoro-α-D-galactosyl fluoride to trap an intermediate on green coffee beanα-galactosidase and identify the catalytic nucleophile[J].Carbohydrate Research,2000,329(3):539-547.
[2]Van den Ende W.Multifunctional fructans and raffinose family oligosaccharides[J].Frontiers in Plant Science,2013,4(4):247.DOI.org/10.3389/fpls.2013.00247.
[3]周英彪,田朝玉,朱玥明等.在酿酒酵母中重构棉子糖生物合成途径的基础研究[J].现代食品科技,2017,33(5):121-128.
[4]Adamberg K,Adamberg S,Ernits K,et al.Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose,raffinose and raffinose-derived fructans[J].Anaerobe,2018,52(8):100-110.
[5]朱庆莉,兰宏兵,云志.双液相溶剂浸出棉籽中棉子糖和棉酚的工艺[J].南京工业大学学报:自然科学版,2014,36(4):114-117.
[6]李芳,汪晓峰.植物中棉子糖系列寡糖代谢及其调控关键酶研究进展[J].西北植物学报,2008,28(4):852-859.
[7]Taji T,Ohsumi C,Iuchi S,et al.Important roles of drought-and cold-inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[J].The Plant Journal,2002,29(4):417-426.
[8]Egert A,Keller F,Peters S.Abiotic stress-induced accumulation of raffinose in Arabidopsis leaves is mediated by a single raffinose synthase(RS5,At5g40390)[J].BMC Plant Biology,2013,13(1):218.DOI.org/10.1186/1471-2229-13-218.
[9]Gangola M P,Jaiswal S,Kannan U,et al.Galactinol synthase enzyme activity influences raffinose family oligosaccharides(RFO)accumulation in developing chickpea(Cicer arietinum L.)seeds[J].Phytochemistry,2016,125:88-98.
[10]Kannan U,Sharma R,Khedikar Y,et al.Differential expression of two galactinol synthase isoforms Lc GolS1 and Lc GolS2 in developing lentil(Lens culinaris Medik.cv CDC Redberry)seeds[J].Plant Physiology and Biochemistry,2016,108:422-433.
[11]谷雷.玉米肌醇半乳糖苷合成酶2基因(Zm GOLS2)的功能和表达调控研究[D].咸阳:西北农林科技大学,2018.
[12]Maruyama K,Urano K,Yoshiwara K,et al.Integrated analysis of the effects of cold and dehydration on rice metabolites,phytohormones,and gene transcripts[J].Plant Physiology,2014,164(4):1759-1771.
[13]乔梦,田原,杨瑞,等.小麦棉子糖合成酶基因(TaRS)的克隆及特性分析[J].农业生物技术学报,2017,25(8):1245-1254.
[14]左静.梅花肌醇半乳糖苷和棉子糖合成酶基因的克隆与功能初探[D].武汉:华中农业大学,2017.
[15]眭晓蕾.黄瓜棉子糖合成酶基因克隆、功能验证及其低温响应研究[C].中国园艺学会2011年学术年会论文集:中国园艺学会,2011:1.
[16]张瑞腾,吕建春,周梦迪等.甜瓜肌醇半乳糖苷合成酶基因CmGAS1的表达与功能分析[J].园艺学报,2018,45(10):1929-1940.
[17]冯睿杰,侯丽霞,郭扬等.葡萄肌醇半乳糖苷合成酶基因Vv GolS2-4的克隆及表达特性分析[J].华北农学报,2017,32(02):87-95.
[18]王毅,肖良俊,马婷等.泡核桃肌醇半乳糖苷合成酶基因克隆及表达分析[J].基因组学与应用生物学,2018,37(05):2029-2033.
[19]李娜.辣椒肌醇半乳糖苷合成酶基因克隆与低温表达分析[D].扬州:扬州大学,2008.
[20]王君珂.基于RNA-Seq分析甜菜碱和棉子糖代谢途径在小桐子干旱响应与适应过程中的作用[D].昆明:云南师范大学,2017.
[21]George S,Manoharan D,Li J,et al.Drought and salt stress in Macrotyloma uniflorum leads to common and specific transcriptomic responses and reveals importance of raffinose family oligosaccharides in stress tolerance[J].Gene Reports,2018,10:7-16.
[22]Jing Y,Lang S,Wang D,et al.Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds[J].Journal of Plant Physiology,2018,230:109-121.
[23]Tian C,Yang J,Zeng Y,et al.Biosynthesis of raffinose and stachyose from sucrose via an in vitro multienzyme system[J].Appl.Environ.Microbiol.,2018:AEM.02306-18.DOI:10.1128/AEM.02306-18.
[24]Pharr D M,Sox H N,Locy R D,et al.Partial characterization of the galactinol forming enzyme from leaves of Cucumis sativus L[J].Plant Science Letters,1981,23(1):25-33.
[25]Handlet L W,Pharr D M.Ion stimulation,UDP inhibition and effects of sulfhydryl reagents on the activity of galactinol synthase from leaves of cucumber,Cucumis sativus L[J].Zeitschrift für Pflanzenphysiologie,1982,108(5):447-455.
[26]Smith P T,Kuo T M,Crawford C G.Purification and characterization of galactinol synthase from mature zucchini squash leaves[J].Plant Physiology,1991,96(3):693-698.
[27]Hart D O,He S,Chany C J,et al.Identification of Asp-130 as the catalytic nucleophile in the mainα-galactosidase from Phanerochaete chrysosporium,a family 27 glycosyl hydrolase[J].Biochemistry,2000,39(32):9826-9836.
[28]Ly H D,Howard S,Shum K,et al.The synthesis,testing and use of 5-fluoro-α-D-galactosyl fluoride to trap an intermediate on green coffee beanα-galactosidase and identify the catalytic nucleophile[J].Carbohydrate Research,2000,329(3):539-547.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |