面包树果实凝集素的提取纯化及理化性质分析
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摘要
建立面包树果实凝集素(frutalin,FTL)的提取纯化方案,并对FTL纯品进行部分理化性质分析。本试验以新鲜的面包树果实籽为材料,经Tris-HCl缓冲液浸提、过滤、硫酸铵沉淀和阳离子交换层析后超滤浓缩得FTL纯品,最后用纯品开展了糖抑制,热稳定性,pH稳定性和金属离子耐受性等试验。提取优化得到的最佳条件为:料液比1∶2(g:m L),浸提时间2 h,浸提温度50℃,硫酸铵饱和度70%,沉淀时间4 h。经阳离子交换层析后FTL回收率为22%。研究结果表明,FTL是一种热稳定性凝集素,在温度不超过60℃时,其凝血活性不受影响,并且不依赖于金属离子来发挥其凝血活性。本研究建立了一个简单高效的FTL分离纯化方案,并对FTL纯品做了部分理化性质研究,为FTL可能的生物医学应用提供研究基础。
To establish the extraction and purification process of frutalin from Artocarpus incisa(breadfruit) seeds,and then analyze the physicochemical properties of the purified frutalin. In this experiment,Artocarpus incisa(breadfruit) seeds were used as materials. The crude frutalin was extracted from fresh breadfruit seeds with Tris-HCl buffer and precipitated with ammonium sulfate,which was further purified with cation exchange chromatography and ultrafiltration. After purified frutalin obtained,several physicochemical properties,including sugar inhibition,thermostability,pH stability and metal ion tolerance,were tested. The optimal extraction condition of frutalin was obtained as follows:the ratio of material to liquid was 1∶ 2(g∶m L),the extraction time was 2 h,the extraction temperature was 50 ℃,the saturation of ammonium sulfate was 70% and the precipitation time is 4 h. The yield of purified frutalin was 22%. The results show that FTL is a kind of thermostable lectin,and its haemagglutinating activity will not damage when the temperature keep no high than 60 ℃,and the haemagglutinating activity does not effected by common metal ions. In a word,a simple and effective separation and purification protocol of frutalin has been set up,followed with a series of physicochemical properties analysis with the purified frutalin. All mentioned above results may pave the way for the biomedical application of frutalin.
To establish the extraction and purification process of frutalin from Artocarpus incisa(breadfruit) seeds,and then analyze the physicochemical properties of the purified frutalin. In this experiment,Artocarpus incisa(breadfruit) seeds were used as materials. The crude frutalin was extracted from fresh breadfruit seeds with Tris-HCl buffer and precipitated with ammonium sulfate,which was further purified with cation exchange chromatography and ultrafiltration. After purified frutalin obtained,several physicochemical properties,including sugar inhibition,thermostability,pH stability and metal ion tolerance,were tested. The optimal extraction condition of frutalin was obtained as follows:the ratio of material to liquid was 1∶ 2(g∶m L),the extraction time was 2 h,the extraction temperature was 50 ℃,the saturation of ammonium sulfate was 70% and the precipitation time is 4 h. The yield of purified frutalin was 22%. The results show that FTL is a kind of thermostable lectin,and its haemagglutinating activity will not damage when the temperature keep no high than 60 ℃,and the haemagglutinating activity does not effected by common metal ions. In a word,a simple and effective separation and purification protocol of frutalin has been set up,followed with a series of physicochemical properties analysis with the purified frutalin. All mentioned above results may pave the way for the biomedical application of frutalin.
引文
1 Tan HQ,Zhang CC,Qu X,et al.Application of plant agglutinin in biomedical field[J].Chin Polym Bull(高分子通报),2016,9:91-102.
2 Bao JK.Plant lectins:multifunctions and applications[J].Chin Bull of Life Sci(生命科学),2011,23:533-540.
3 Jiang K,Li W,Zhang Q,et al.GP73 N-glycosylation at Asn144 reduces hepatocellular carcinoma cell motility and invasiveness[J].Oncotarget,2016,7:23530-23541.
4 Norton P,Comunale MA,Herrera H,et al.Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma[J].Proteomics,2016,16:3126-3136.
5 Pei LL,Xiao CQ,He W.Application of lectin chip technology in diagnosis of common gynecological tumors[J].Acta Univ Med Anhui(安徽医科大学学报),2013,48:1348-1351.
6 Akkouh O,Ng TB,Singh SS,et al.Lectins with anti-HIV activity:a review[J].Molecules,2015,20:648-668.
7 Xu XC,Zhang ZW,Chen YE,et al.Antiviral and antitumor activities of the lectin extracted from Aspidistra elatior[J].ZNaturforsch C,2015,70(1-2):7-13.
8 Muslim SN.Improving of antibacterial activity for antibiotics by purified and characterized lectin from acinetobacter baumannii[J].Iraqi J Biotechnol,2015,14(1):9-22.
9 Oliveira C,Costa S,Teixeira JA,et al.c DNA cloning and functional expression of the alpha-D-galactose-binding lectin frutalin in Escherichia coli[J].Mol Biotechnol,2009,43:212-220.
10 Moreira RA,Castelo-Branco CC,Monteiro ACO,et al.Isolation and partial characterization of a lectin from Artocarpus incisa L.seeds[J].Phytochemistry,1998,47:1183-1188.
11 Oliveira C,Teixeira JA,Schmitt F,et al.A comparative study of recombinant and native frutalin binding to human prostate tissues[J].Bmc Biotechnology,2009,9(1):1-7.
12 Ana COM,Humberto DMP,Antonio EVN,et al.Crystallization and preliminary X-ray diffraction studies of frutalin,anα-D-galactose-specific lectin from Artocarpus incisa seeds[J].Acta Crystallographica,2015,71:1282-1285.
13 Oliveira C,Nicolau A,Teixeira JA,et al.Cytotoxic effects of native and recombinant frutalin,a plant galactose-binding lectin,on He La cervical cancer cells[J].J Biomed Biotechnol,2011,2011:568932-568940.
14 Oliveira C,Teixeira JA,Domingues L.Recombinant production of plant lectins in microbial systems for biomedical application-the frutalin case study[J].Front Plant Sci,2014,5:390-395.
15 Tang HS,Ning XF,Cheng LL,et al.Preliminary study of the activity of the crude lectin from Peganum harmala seeds[J].Acta Bot.Bor-Occid Sin(西北植物学报),2010,30:813-820.
16 Chen G,Wang HB,Guo R,et al.Extraction,separation and purification of Phaseolus vulgaris agglutinin(PHA)from common bean fresh pods[J].J Jianghan Univ Sin(江汉大学学报),2016,44:496-503.
17 Deng ZD,Cheng AF,Li XL.Study on the extraction and agglutination of lectin from Melastoma dodecandrum Lour[J].Heilongjiang Agric Sci(黑龙江农业科学),2015,2:56-58.
18 Liu PJ,Fu ZH,Sun D,et al.Separation and purification of lectin from Morus alba L.and its antimicrobial activity[J].Nat Prod Res Dev(天然产物研究与开发),2015,27:475-479.
19 Chen XH,Ren JH,Wei YM,et al.Optimization of extraction technology of an immune active protein from astragali radix[J].Chin J Exp Tradit Med Form(中国实验方剂学杂志),2016,5:13-17.
2 Bao JK.Plant lectins:multifunctions and applications[J].Chin Bull of Life Sci(生命科学),2011,23:533-540.
3 Jiang K,Li W,Zhang Q,et al.GP73 N-glycosylation at Asn144 reduces hepatocellular carcinoma cell motility and invasiveness[J].Oncotarget,2016,7:23530-23541.
4 Norton P,Comunale MA,Herrera H,et al.Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma[J].Proteomics,2016,16:3126-3136.
5 Pei LL,Xiao CQ,He W.Application of lectin chip technology in diagnosis of common gynecological tumors[J].Acta Univ Med Anhui(安徽医科大学学报),2013,48:1348-1351.
6 Akkouh O,Ng TB,Singh SS,et al.Lectins with anti-HIV activity:a review[J].Molecules,2015,20:648-668.
7 Xu XC,Zhang ZW,Chen YE,et al.Antiviral and antitumor activities of the lectin extracted from Aspidistra elatior[J].ZNaturforsch C,2015,70(1-2):7-13.
8 Muslim SN.Improving of antibacterial activity for antibiotics by purified and characterized lectin from acinetobacter baumannii[J].Iraqi J Biotechnol,2015,14(1):9-22.
9 Oliveira C,Costa S,Teixeira JA,et al.c DNA cloning and functional expression of the alpha-D-galactose-binding lectin frutalin in Escherichia coli[J].Mol Biotechnol,2009,43:212-220.
10 Moreira RA,Castelo-Branco CC,Monteiro ACO,et al.Isolation and partial characterization of a lectin from Artocarpus incisa L.seeds[J].Phytochemistry,1998,47:1183-1188.
11 Oliveira C,Teixeira JA,Schmitt F,et al.A comparative study of recombinant and native frutalin binding to human prostate tissues[J].Bmc Biotechnology,2009,9(1):1-7.
12 Ana COM,Humberto DMP,Antonio EVN,et al.Crystallization and preliminary X-ray diffraction studies of frutalin,anα-D-galactose-specific lectin from Artocarpus incisa seeds[J].Acta Crystallographica,2015,71:1282-1285.
13 Oliveira C,Nicolau A,Teixeira JA,et al.Cytotoxic effects of native and recombinant frutalin,a plant galactose-binding lectin,on He La cervical cancer cells[J].J Biomed Biotechnol,2011,2011:568932-568940.
14 Oliveira C,Teixeira JA,Domingues L.Recombinant production of plant lectins in microbial systems for biomedical application-the frutalin case study[J].Front Plant Sci,2014,5:390-395.
15 Tang HS,Ning XF,Cheng LL,et al.Preliminary study of the activity of the crude lectin from Peganum harmala seeds[J].Acta Bot.Bor-Occid Sin(西北植物学报),2010,30:813-820.
16 Chen G,Wang HB,Guo R,et al.Extraction,separation and purification of Phaseolus vulgaris agglutinin(PHA)from common bean fresh pods[J].J Jianghan Univ Sin(江汉大学学报),2016,44:496-503.
17 Deng ZD,Cheng AF,Li XL.Study on the extraction and agglutination of lectin from Melastoma dodecandrum Lour[J].Heilongjiang Agric Sci(黑龙江农业科学),2015,2:56-58.
18 Liu PJ,Fu ZH,Sun D,et al.Separation and purification of lectin from Morus alba L.and its antimicrobial activity[J].Nat Prod Res Dev(天然产物研究与开发),2015,27:475-479.
19 Chen XH,Ren JH,Wei YM,et al.Optimization of extraction technology of an immune active protein from astragali radix[J].Chin J Exp Tradit Med Form(中国实验方剂学杂志),2016,5:13-17.