三色雷蘑核糖核酸酶的分离纯化和理化性质
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摘要
依次采用离子交换层析(DEAE-Cellulose、CM-Cellulose和SP-Sepharose)和凝胶过滤层析(Superdex75 HR10/300 GL)从三色雷蘑(Leucopaxillus tricolor)子实体中分离纯化得到一种核糖核酸酶(Ribonuclease,RNase)。该酶比活为1406 U/mg,纯化倍数为154,通过HPLC-LTQ-Orbitrap测序得到4条肽段序列,分别为DMAVSYLSR、IGLSSIPR、ILGRFVVDTYK和SGKANAATPK。该核糖核酸酶的相对分子质量为15000,最适反应pH为9.0,最适反应温度为40℃,热稳定性高,80℃孵育1 h后能保持90%以上的活性:Cu~(2+)、Ca~(2+)、Mn~(2+)、Cd~(2+)和Al~(3+)在1.25～10 mmol/L浓度下对该酶活性具有抑制作用,且抑制作用随着金属离子浓度的升高而变强;Fe~(2+)在1.25～5 mmol/L的浓度下,Pb~(2+)、Zn~(2+)、Mg~(2+)、Hg~(2+)、Fe~(3+)在1.25～2.5 mmol/L的浓度下对该酶的活性有促进作用。
A ribonuclease(RNase)was purified from Leucopaxillus tricolor fruit bodies by ion exchange chromatography(DEAE-Cellulose,CM-Cellulose and SP-Sepharose)and gel filtration(Superdex 75 HR10/300 GL).The purification fold of this RNase was 154 and the specific activity was 1406 U/mg.Four amino acid sequences were obtained(DMAVSYLSR,IGLSSIPR,ILGRFVVDTYK and SGKANAATPK)by HPLC-LTQOrbitrap identification of the RNase.The relative molecular mass of the RNase was estimated to be 15000.The enzyme displayed pH and temperature optima of 9.0 and 40 ℃,respectively.The enzyme showed good thermal stability and could remain more than 90% of the activity after 1 h exposure to 80 ℃.The enzyme activity was inhibited by Cu~(2+),Ca~(2+),Mn~(2+),Cd~(2+) and Al~(3+) at concentration of 1.25～10 mmol/L and the inhibition effect increased with the increase of metal ion concentration.The enzyme activity was stimulated by Fe~(2+) at concentration of 1.25～5 mmol/L and Pb~(2+),Zn~(2+),Mg~(2+),Hg~(2+),Fe~(3+) at concentration of 1.25～2.5 mmol/L.
A ribonuclease(RNase)was purified from Leucopaxillus tricolor fruit bodies by ion exchange chromatography(DEAE-Cellulose,CM-Cellulose and SP-Sepharose)and gel filtration(Superdex 75 HR10/300 GL).The purification fold of this RNase was 154 and the specific activity was 1406 U/mg.Four amino acid sequences were obtained(DMAVSYLSR,IGLSSIPR,ILGRFVVDTYK and SGKANAATPK)by HPLC-LTQOrbitrap identification of the RNase.The relative molecular mass of the RNase was estimated to be 15000.The enzyme displayed pH and temperature optima of 9.0 and 40 ℃,respectively.The enzyme showed good thermal stability and could remain more than 90% of the activity after 1 h exposure to 80 ℃.The enzyme activity was inhibited by Cu~(2+),Ca~(2+),Mn~(2+),Cd~(2+) and Al~(3+) at concentration of 1.25～10 mmol/L and the inhibition effect increased with the increase of metal ion concentration.The enzyme activity was stimulated by Fe~(2+) at concentration of 1.25～5 mmol/L and Pb~(2+),Zn~(2+),Mg~(2+),Hg~(2+),Fe~(3+) at concentration of 1.25～2.5 mmol/L.
引文
[1]SCHUSTER C,GLASE R,FIALA C,et al.Prenatal human skin expresses the antimicrobial peptide RNase 7[J].Archives of Dermatological Research,2013,305(6):545-549.
[2]ANDERSON K,DUNMAN P.Messenger RNA turnover processes in Escherichia coli,Bacillus subtilis,and emerging studies in Staphylococcus aureus[J].International Journal of Microbiology,2009(1):525491.
[3]徐丽婧.粉紫香蘑核糖核酸酶和草菇植酸酶的纯化及双孢蘑菇Horst U1重组的研究[D].北京:中国农业大学,2012.
[4]吴莹莹.紫丁香蘑蛋白酶、浅黄褐乳菇凝集素和核糖核酸酶的分离纯化及理化性质研究[D].北京:中国农业大学,2012.
[5]耿雪冉.松茸和三色雷蘑血管紧张素转换酶抑制剂的分离纯化、结构及生物活性研究[D].北京:中国农业大学,2016.
[6]GENG X,TIAN GT,ZHANG WW,et al.Isolation of an angiotensin I-converting enzyme inhibitory protein with antihypertensive effect in spontaneously hypertensive rats from the edible wild mushroom Leucopaxillus tricolor[J].Molecules,2015,20(6):10141-10153.
[7]GENG X,FAN J,XU L,et al.Hydrolysis of oligosaccharides by a fungal alpha-galactosidase from fruiting bodies of a wild mushroom Leucopaxillus tricolor[J].Journal of Basic Microbiology,2018,58(12):1043-1052.
[8]耿雪冉,柳万石,王贺祥.污白干酪菌核糖核酸酶的分离纯化和理化性质[J].中国食用菌,2015,34(6):63-66.
[9]张哲,牛万宝,赵靖坤,等.白林地蘑菇核糖核酸酶的分离纯化及理化性质研究[J].食用菌,2009,31(6):8-9.
[10]WANG H,NG T.A novel ribonuclease from fresh fruiting bodies of the portabella mushroom Agaricus bisporus[J].Biochemistry and Cell Biology,2006,84(2):178-183.
[11]WANG H,NG T.A new ribonuclease from the black oyster mushroom Pleurotus ostreatus[J].Peptides,2004,25(4):685-687.
[12]NG T,WANG H.A novel ribonuclease from fruiting bodies of the common edible mushroom Pleurotus eryngii[J].Peptides,2004,25(8):1365-1368.
[13]WANG H,NG T.Isolation of a new ribonuclease from fresh fruiting bodies of the straw mushroom[J].Biochemical and Biophysical Research Communications,1999,264(3):714-718.
[14]WANG H,NG T,CHIU S.A distinctive ribonuclease from fresh druiting bodies of the medicinal mushroom Ganoderma lucidum[J].Biochemical and Biophysical Research Communications,2004,314(2):519-522.
[15]WANG H,NG T.Purification of a novel ribonuclease from dried fruiting bodies of the edible wild mushroom Thelephora ganbajun[J].Biochemical and Biophysical Research Communications,2004,324(2):855-859.
[16]陈晓.印度块菌核糖核酸酶的分离纯化及美味牛肝菌降低泡菜中亚硝酸盐的研究[D].北京:中国农业大学,2013.
[17]WANG H,NG T.A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens[J].Biochemical and Biophysical Research Communications,2003,312(4):965-968.
[18]崔黎.金顶侧耳蛋白酶、核糖核酸酶与桃红侧耳核糖核酸酶的纯化及生化特性研究[D].北京:中国农业大学,2008.
[19]WANG H,NG T.Isolation of a ribonuclease from fruiting bodies of the wild mushroom Termitomyces globulus[J].Peptides,2003,24(7):973-977.
[20]WANG H,NG T.A novel ribonuclease from the veiled lady mushroom Dictyophora indusiata[J].Biochemistry and Cell Biology,2003,81(6):373-377.
[21]马增强.隐孔菌抗PRRSV生理活性物质及保肝作用的研究[D].北京:中国农业大学,2014.
[2]ANDERSON K,DUNMAN P.Messenger RNA turnover processes in Escherichia coli,Bacillus subtilis,and emerging studies in Staphylococcus aureus[J].International Journal of Microbiology,2009(1):525491.
[3]徐丽婧.粉紫香蘑核糖核酸酶和草菇植酸酶的纯化及双孢蘑菇Horst U1重组的研究[D].北京:中国农业大学,2012.
[4]吴莹莹.紫丁香蘑蛋白酶、浅黄褐乳菇凝集素和核糖核酸酶的分离纯化及理化性质研究[D].北京:中国农业大学,2012.
[5]耿雪冉.松茸和三色雷蘑血管紧张素转换酶抑制剂的分离纯化、结构及生物活性研究[D].北京:中国农业大学,2016.
[6]GENG X,TIAN GT,ZHANG WW,et al.Isolation of an angiotensin I-converting enzyme inhibitory protein with antihypertensive effect in spontaneously hypertensive rats from the edible wild mushroom Leucopaxillus tricolor[J].Molecules,2015,20(6):10141-10153.
[7]GENG X,FAN J,XU L,et al.Hydrolysis of oligosaccharides by a fungal alpha-galactosidase from fruiting bodies of a wild mushroom Leucopaxillus tricolor[J].Journal of Basic Microbiology,2018,58(12):1043-1052.
[8]耿雪冉,柳万石,王贺祥.污白干酪菌核糖核酸酶的分离纯化和理化性质[J].中国食用菌,2015,34(6):63-66.
[9]张哲,牛万宝,赵靖坤,等.白林地蘑菇核糖核酸酶的分离纯化及理化性质研究[J].食用菌,2009,31(6):8-9.
[10]WANG H,NG T.A novel ribonuclease from fresh fruiting bodies of the portabella mushroom Agaricus bisporus[J].Biochemistry and Cell Biology,2006,84(2):178-183.
[11]WANG H,NG T.A new ribonuclease from the black oyster mushroom Pleurotus ostreatus[J].Peptides,2004,25(4):685-687.
[12]NG T,WANG H.A novel ribonuclease from fruiting bodies of the common edible mushroom Pleurotus eryngii[J].Peptides,2004,25(8):1365-1368.
[13]WANG H,NG T.Isolation of a new ribonuclease from fresh fruiting bodies of the straw mushroom[J].Biochemical and Biophysical Research Communications,1999,264(3):714-718.
[14]WANG H,NG T,CHIU S.A distinctive ribonuclease from fresh druiting bodies of the medicinal mushroom Ganoderma lucidum[J].Biochemical and Biophysical Research Communications,2004,314(2):519-522.
[15]WANG H,NG T.Purification of a novel ribonuclease from dried fruiting bodies of the edible wild mushroom Thelephora ganbajun[J].Biochemical and Biophysical Research Communications,2004,324(2):855-859.
[16]陈晓.印度块菌核糖核酸酶的分离纯化及美味牛肝菌降低泡菜中亚硝酸盐的研究[D].北京:中国农业大学,2013.
[17]WANG H,NG T.A ribonuclease with distinctive features from the wild green-headed mushroom Russulus virescens[J].Biochemical and Biophysical Research Communications,2003,312(4):965-968.
[18]崔黎.金顶侧耳蛋白酶、核糖核酸酶与桃红侧耳核糖核酸酶的纯化及生化特性研究[D].北京:中国农业大学,2008.
[19]WANG H,NG T.Isolation of a ribonuclease from fruiting bodies of the wild mushroom Termitomyces globulus[J].Peptides,2003,24(7):973-977.
[20]WANG H,NG T.A novel ribonuclease from the veiled lady mushroom Dictyophora indusiata[J].Biochemistry and Cell Biology,2003,81(6):373-377.
[21]马增强.隐孔菌抗PRRSV生理活性物质及保肝作用的研究[D].北京:中国农业大学,2014.