汽巴蓝F3GA染料亲和色谱介质的制备及其对黑芸豆凝集素的特异性吸附
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摘要
采用反相悬浮再生法制备6 g/100 mL的琼脂糖微球,采用环氧氯丙烷对琼脂糖微球进行二次交联,并进行染料配体汽巴蓝F3GA的偶联,得到一种新型活性Blue Beads 6FF微球。通过傅里叶变换红外光谱、扫描电子显微镜、压力流速曲线、溶胀率、泄漏率和孔隙率等对微球物理性能进行表征。结果发现,微球球形度较好,粒径大小均一,平均粒径为56.37μm左右,具有较高的孔隙率、溶胀率及广范围的pH值稳定性,且耐压能力强。以黑芸豆凝集素为目标蛋白,通过Langmuir吸附等温式拟合,结果表明该填料对黑芸豆蛋白有较高的吸附量(19.7 mg/mL),微球吸附性能良好,可实现对凝集素蛋白的快速分离和纯化。
Agarose microspheres(6 g/100 mL) were prepared by reversed-phase suspension regeneration method and they were cross-linked twice with epichlorohydrin and then coupled with the dye Cibacron Blue F3 GA as a special ligand to form novel active microspheres(Blue Beads 6 FF). The properties of the dye affinity medium were characterized by Fourier transform infrared(FTIR) spectroscopy, scanning electron microscopy(SEM), pressure against flow velocity curve, swelling rate, leakage rate, and porosity. Our results showed that the microspheres had a good sphericity and uniform size with high porosity and swelling rate, a wide range of pH stability and strong pressure resistance. The average particle size of the microspheres was approximately 56.37 μm. The adsorption properties of the dye affinity medium were evaluated for lectin purification from black kidney beans. By fitting the typical Langmuir adsorption isotherm, the equilibrium adsorption capacity of lectin from black kidney beans was determined as 19.7 mg/mL microspheres. The dye affinity microspheres enabled rapid isolation and purification of lectin.
Agarose microspheres(6 g/100 mL) were prepared by reversed-phase suspension regeneration method and they were cross-linked twice with epichlorohydrin and then coupled with the dye Cibacron Blue F3 GA as a special ligand to form novel active microspheres(Blue Beads 6 FF). The properties of the dye affinity medium were characterized by Fourier transform infrared(FTIR) spectroscopy, scanning electron microscopy(SEM), pressure against flow velocity curve, swelling rate, leakage rate, and porosity. Our results showed that the microspheres had a good sphericity and uniform size with high porosity and swelling rate, a wide range of pH stability and strong pressure resistance. The average particle size of the microspheres was approximately 56.37 μm. The adsorption properties of the dye affinity medium were evaluated for lectin purification from black kidney beans. By fitting the typical Langmuir adsorption isotherm, the equilibrium adsorption capacity of lectin from black kidney beans was determined as 19.7 mg/mL microspheres. The dye affinity microspheres enabled rapid isolation and purification of lectin.
引文
[1]ZHANG J,SHI J,SANJA I,et al.Biological properties and characterization of lectin from red kidney bean(Phaseolus vulgaris)[J].Food Reviews International,2008,25(1):12-27.DOI:10.1080/87559120802458115.
[2]KUMAR S,VERMA A K,DAS M,et al.Clinical complications of kidney bean(Phaseolus vulgaris L.)consumption[J].Nutrition,2013,29(6):821-827.DOI:10.1016/j.nut.2012.11.010.
[3]BUUL V J V,BROUNS F J P H.Health effects of wheat lectins:a review[J].Journal of Cereal Science,2014,59(2):112-117.DOI:10.1016/j.jcs.2014.01.010.
[4]HE S,SIMPSON B K,NGADI M O,et al.In vitro studies of the digestibility of lectin from black turtle bean(Phaseolus vulgaris)[J].Food Chemistry,2015,173(1):397-404.DOI:10.1016/j.foodchem.2014.10.045.
[5]HE S,SIMPSON B K,SUN H,et al.Phaseolus vulgaris lectins:a systematic review of characteristics and health implications[J].Critical Reviews in Food Science&Nutrition,2018,58(1):70-83.DOI:10.1080/10408398.2015.1096234.
[6]NASCIMENTO K S,CUNHA A I,NASCIMENTO K S,et al.An overview of lectins purification strategies[J].Journal of Molecular Recognition,2012,25(11):527-541.DOI:10.1002/jmr.2200.
[7]HE Shudong,SHI J,WALID E,et al.Reverse micellar extraction of lectin from black turtle bean(Phaseolus vulgaris):optimisation of extraction conditions by response surface methodology[J].Food Chemi s t ry,2015,166(1):93-100.DOI:10.1016/j.foodchem.2014.05.156.
[8]DENIZLI A,PI狵IN E.Dye-ligand affinity systems[J].Journal of Biochemical and Biophysical Methods,2001,49(1):391-416.DOI:10.1016/S0165-022X(01)00209-3.
[9]ARORA S,SAXENA V,AYYAR B V.Affinity chromatography:a versatile technique for antibody purification[J].Methods,2016,116(1):84-94.DOI:10.1016/j.ymeth.2016.12.010.
[10]王冬宇,郑喜群.ConA亲和层析法在纯化糖蛋白中的应用[J].齐齐哈尔大学学报(自然科学版),2016,32(5):46-49.DOI:10.3969/j.issn.1007-984X.2016.05.012.
[11]SINTIPRUNGRAT K,CHAISURIYA P,WATCHARATANYATIPK,et al.Immunoaffinity chromatography in antivenomics studies:various parameters that can affect the results[J].Toxicon,2016,119(1):129-139.DOI:10.1016/j.toxicon.2016.05.017.
[12]王利民,秦贵信,刘林娜,等.三种亲和层析体系纯化大豆凝集素的比较研究[J].大豆科学,2007,26(1):51-54.DOI:10.3969/j.issn.1000-9841.2007.01.012.
[13]张海涛,聂华丽,陈天翔,等.木瓜蛋白酶在染料Cibacron Blue F3GA纤维素亲和膜上的吸附研究[J].化学工程,2009,37(7):38-41;66.DOI:10.3969/j.issn.1005-9954.2009.07.010.
[14]申屠静灵,贾之慎,高慧.活性蓝F3GA固载的壳聚糖微球对过氧化氢酶的吸附[J].浙江大学学报(理学版),2005,32(6):674-677;684.DOI:10.3321/j.issn:1008-9497.2005.06.018.
[15]SUBRAMANIAN S.Dye-ligand affinity chromatography:the interaction of Cibacron Blue F3GA with proteins and enzymes[J].Crc Critical Reviews in Biochemistry,1984,16(2):169-205.DOI:10.3109/10409238409102302.
[16]HE Shudong,SHI J,WALID E,et al.Extraction and purification of a lectin from small black kidney bean(Phaseolus vulgaris)using a reversed micellar system[J].Process Biochemistry,2013,48(4):746-752.DOI:10.1016/j.procbio.2013.02.007.
[17]尹霜霜,屠丹宇,徐陈燕,等.磁性琼脂糖微球的制备及其蛋白吸附性能研究[J].湖州师范学院学报,2016,38(8):59-64.DOI:10.3969/j.issn.1009-1734.2016.08.010.
[18]杨晓玲,张振,朱以华.多孔高胺基交联壳聚糖微球的制备和对有机染料吸附性能的研究[J].过程工程学报,2009,9(增刊2):31-34.
[19]卜春苗,王有贤,龚波林,等.活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用[J].色谱,2008,26(3):378-383.DOI:10.3321/j.issn:1000-8713.2008.03.024.
[20]HUANG Yongdong,ZHANG Rongyue,LI Juan,et al.Anovel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins[J].Protein Expression and Purification,2014,95(1):84-91.DOI:10.1016/j.pep.2013.11.004.
[21]ZHANG Lina,ZHOU Jinping,YANG Guang,et al.Preparative fractionation of polysaccharides by columns packed with regenerated cellulose gels[J].Journal of Chromatography A,1998,816(2):131-136.DOI:10.1016/S0021-9673(98)00475-0.
[22]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,22(7):680-685.DOI:10.1038/227680a0.
[23]黄广诚,邱比特,刘亚栋,等.粒径和孔径可控的多孔交联聚苯乙烯微球的制备[J].应用化学,2016,33(4):406-411.DOI:10.11944/j.issn.1000-0518.2016.04.150312.
[24]周青竹,马光辉,苏志国.尺寸均一的琼脂糖层析介质的制备[C].全国化学工程与生物化工年会,北京:中国化工学会,2005.
[25]隋郁,郭晓霞,印杰,等.高Tg可溶含偶氮三嗪染料基团的侧链型共聚聚酰亚胺的合成与表征[J].高等学校化学学报,2000,21(12):1944-1947.DOI:10.3321/j.issn:0251-0790.2000.12.028.
[26]王阳木,闭静秀,赵岚,等.一种新型的琼脂糖疏水层析介质开发及其在重组乙肝表面抗原(CHO-HBsAg)分离纯化中的应用[J].生物工程学报,2006,22(2):278-284.DOI:10.13345/j.cjb.2006.02.020.
[27]张静,张政朴,宋瑜,等.染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究[J].高等学校化学学报,2005,26(12):2363-2368;2195.DOI:10.3321/j.issn:0251-0790.2005.12.031.
[28]CHU K T,NG T B.Purification and characterization of a chitinaselike antifungal protein from black turtle bean with stimulatory effect on nitric oxide production by macrophages[J].Biological Chemistry,2005,289(1):19-24.DOI:10.1515/BC.2005.003.
[29]HE S,SIMPSON B K,NGADI M O,et al.pH stability study of lectin from black turtle bean(Phaseolus vulgaris)as influenced by guanidinium-HCl and thermal treatment[J].Protein&Peptide Letters,2015,22(1):45-51.DOI:10.2174/0929866521666140909155556.
[30]王启迪,罗坚,全灿,等.蛋白质纯化技术研究进展[J].现代生物医学进展,2017,17(32):6389-6392.DOI:10.13241/j.cnki.pmb.2017.32.042.
[31]肖秀情,丁学知,夏立秋,等.藠头凝集素的分离纯化和凝集活性分析[J].食品科学,2013,34(19):80-83.DOI:10.7506/spkx1002-6630-201319018.
[32]REN Jiaoyan,SHI J,YUKIO K,et al.Comparison of the phytohaemagglutinin from red kidney bean(Phaseolus vulgaris)purified by different affinity chromatography[J].Food Chemistry,2008,108(1):394-401.DOI:10.1016/j.foodchem.2007.10.071.
[33]DOGN A,OKARA S,SARI M M,et al.Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system[J].Journal of Chromatography B,2012,893-894(8):69-76.DOI:10.1016/j.jchromb.2012.02.036.
[34]KURALAY F,YILMAZ E,UZUN L,et al.Cibacron Blue F3GAmodified disposable pencil graphite electrode for the investigation of affinity binding to bovine serum albumin[J].Colloids and Surfaces B Biointerfaces,2013,110(10):270-274.DOI:10.1016/j.colsurfb.2013.04.024.
[2]KUMAR S,VERMA A K,DAS M,et al.Clinical complications of kidney bean(Phaseolus vulgaris L.)consumption[J].Nutrition,2013,29(6):821-827.DOI:10.1016/j.nut.2012.11.010.
[3]BUUL V J V,BROUNS F J P H.Health effects of wheat lectins:a review[J].Journal of Cereal Science,2014,59(2):112-117.DOI:10.1016/j.jcs.2014.01.010.
[4]HE S,SIMPSON B K,NGADI M O,et al.In vitro studies of the digestibility of lectin from black turtle bean(Phaseolus vulgaris)[J].Food Chemistry,2015,173(1):397-404.DOI:10.1016/j.foodchem.2014.10.045.
[5]HE S,SIMPSON B K,SUN H,et al.Phaseolus vulgaris lectins:a systematic review of characteristics and health implications[J].Critical Reviews in Food Science&Nutrition,2018,58(1):70-83.DOI:10.1080/10408398.2015.1096234.
[6]NASCIMENTO K S,CUNHA A I,NASCIMENTO K S,et al.An overview of lectins purification strategies[J].Journal of Molecular Recognition,2012,25(11):527-541.DOI:10.1002/jmr.2200.
[7]HE Shudong,SHI J,WALID E,et al.Reverse micellar extraction of lectin from black turtle bean(Phaseolus vulgaris):optimisation of extraction conditions by response surface methodology[J].Food Chemi s t ry,2015,166(1):93-100.DOI:10.1016/j.foodchem.2014.05.156.
[8]DENIZLI A,PI狵IN E.Dye-ligand affinity systems[J].Journal of Biochemical and Biophysical Methods,2001,49(1):391-416.DOI:10.1016/S0165-022X(01)00209-3.
[9]ARORA S,SAXENA V,AYYAR B V.Affinity chromatography:a versatile technique for antibody purification[J].Methods,2016,116(1):84-94.DOI:10.1016/j.ymeth.2016.12.010.
[10]王冬宇,郑喜群.ConA亲和层析法在纯化糖蛋白中的应用[J].齐齐哈尔大学学报(自然科学版),2016,32(5):46-49.DOI:10.3969/j.issn.1007-984X.2016.05.012.
[11]SINTIPRUNGRAT K,CHAISURIYA P,WATCHARATANYATIPK,et al.Immunoaffinity chromatography in antivenomics studies:various parameters that can affect the results[J].Toxicon,2016,119(1):129-139.DOI:10.1016/j.toxicon.2016.05.017.
[12]王利民,秦贵信,刘林娜,等.三种亲和层析体系纯化大豆凝集素的比较研究[J].大豆科学,2007,26(1):51-54.DOI:10.3969/j.issn.1000-9841.2007.01.012.
[13]张海涛,聂华丽,陈天翔,等.木瓜蛋白酶在染料Cibacron Blue F3GA纤维素亲和膜上的吸附研究[J].化学工程,2009,37(7):38-41;66.DOI:10.3969/j.issn.1005-9954.2009.07.010.
[14]申屠静灵,贾之慎,高慧.活性蓝F3GA固载的壳聚糖微球对过氧化氢酶的吸附[J].浙江大学学报(理学版),2005,32(6):674-677;684.DOI:10.3321/j.issn:1008-9497.2005.06.018.
[15]SUBRAMANIAN S.Dye-ligand affinity chromatography:the interaction of Cibacron Blue F3GA with proteins and enzymes[J].Crc Critical Reviews in Biochemistry,1984,16(2):169-205.DOI:10.3109/10409238409102302.
[16]HE Shudong,SHI J,WALID E,et al.Extraction and purification of a lectin from small black kidney bean(Phaseolus vulgaris)using a reversed micellar system[J].Process Biochemistry,2013,48(4):746-752.DOI:10.1016/j.procbio.2013.02.007.
[17]尹霜霜,屠丹宇,徐陈燕,等.磁性琼脂糖微球的制备及其蛋白吸附性能研究[J].湖州师范学院学报,2016,38(8):59-64.DOI:10.3969/j.issn.1009-1734.2016.08.010.
[18]杨晓玲,张振,朱以华.多孔高胺基交联壳聚糖微球的制备和对有机染料吸附性能的研究[J].过程工程学报,2009,9(增刊2):31-34.
[19]卜春苗,王有贤,龚波林,等.活性蓝F3GA固载的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯亲和色谱填料的制备与应用[J].色谱,2008,26(3):378-383.DOI:10.3321/j.issn:1000-8713.2008.03.024.
[20]HUANG Yongdong,ZHANG Rongyue,LI Juan,et al.Anovel gigaporous GSH affinity medium for high-speed affinity chromatography of GST-tagged proteins[J].Protein Expression and Purification,2014,95(1):84-91.DOI:10.1016/j.pep.2013.11.004.
[21]ZHANG Lina,ZHOU Jinping,YANG Guang,et al.Preparative fractionation of polysaccharides by columns packed with regenerated cellulose gels[J].Journal of Chromatography A,1998,816(2):131-136.DOI:10.1016/S0021-9673(98)00475-0.
[22]LAEMMLI U K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,22(7):680-685.DOI:10.1038/227680a0.
[23]黄广诚,邱比特,刘亚栋,等.粒径和孔径可控的多孔交联聚苯乙烯微球的制备[J].应用化学,2016,33(4):406-411.DOI:10.11944/j.issn.1000-0518.2016.04.150312.
[24]周青竹,马光辉,苏志国.尺寸均一的琼脂糖层析介质的制备[C].全国化学工程与生物化工年会,北京:中国化工学会,2005.
[25]隋郁,郭晓霞,印杰,等.高Tg可溶含偶氮三嗪染料基团的侧链型共聚聚酰亚胺的合成与表征[J].高等学校化学学报,2000,21(12):1944-1947.DOI:10.3321/j.issn:0251-0790.2000.12.028.
[26]王阳木,闭静秀,赵岚,等.一种新型的琼脂糖疏水层析介质开发及其在重组乙肝表面抗原(CHO-HBsAg)分离纯化中的应用[J].生物工程学报,2006,22(2):278-284.DOI:10.13345/j.cjb.2006.02.020.
[27]张静,张政朴,宋瑜,等.染料壳聚糖微球的制备及其对牛血清白蛋白(BSA)吸附性能的研究[J].高等学校化学学报,2005,26(12):2363-2368;2195.DOI:10.3321/j.issn:0251-0790.2005.12.031.
[28]CHU K T,NG T B.Purification and characterization of a chitinaselike antifungal protein from black turtle bean with stimulatory effect on nitric oxide production by macrophages[J].Biological Chemistry,2005,289(1):19-24.DOI:10.1515/BC.2005.003.
[29]HE S,SIMPSON B K,NGADI M O,et al.pH stability study of lectin from black turtle bean(Phaseolus vulgaris)as influenced by guanidinium-HCl and thermal treatment[J].Protein&Peptide Letters,2015,22(1):45-51.DOI:10.2174/0929866521666140909155556.
[30]王启迪,罗坚,全灿,等.蛋白质纯化技术研究进展[J].现代生物医学进展,2017,17(32):6389-6392.DOI:10.13241/j.cnki.pmb.2017.32.042.
[31]肖秀情,丁学知,夏立秋,等.藠头凝集素的分离纯化和凝集活性分析[J].食品科学,2013,34(19):80-83.DOI:10.7506/spkx1002-6630-201319018.
[32]REN Jiaoyan,SHI J,YUKIO K,et al.Comparison of the phytohaemagglutinin from red kidney bean(Phaseolus vulgaris)purified by different affinity chromatography[J].Food Chemistry,2008,108(1):394-401.DOI:10.1016/j.foodchem.2007.10.071.
[33]DOGN A,OKARA S,SARI M M,et al.Evaluation of human interferon adsorption performance of Cibacron Blue F3GA attached cryogels and interferon purification by using FPLC system[J].Journal of Chromatography B,2012,893-894(8):69-76.DOI:10.1016/j.jchromb.2012.02.036.
[34]KURALAY F,YILMAZ E,UZUN L,et al.Cibacron Blue F3GAmodified disposable pencil graphite electrode for the investigation of affinity binding to bovine serum albumin[J].Colloids and Surfaces B Biointerfaces,2013,110(10):270-274.DOI:10.1016/j.colsurfb.2013.04.024.