重组人溶菌酶的分离纯化及鉴定
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摘要
目的建立重组人溶菌酶(human lysozyme,h LYZ)的分离纯化方法,并对纯化产物进行鉴定。方法采用固液分离、膜过滤、疏水层析及离子交换层析对hLYZ进行分离纯化;SDS-PAGE及HPLC法检测hLYZ纯度;按《中国药典》二部(2015版)蛋清溶菌酶效价检查法检测其效价;Western blot法检测其特异性;基质辅助激光解吸飞行时间质谱(MALDI-TOF)测定其相对分子质量。结果该分离方法可获得单一组分的重组hLYZ;SDS-PAGE检测纯度达99%,HPLC分析纯度达98. 8%;纯化后的hLYZ可与抗人LYZ抗体特异性结合,活性为1. 2×105 U/mg,相对分子质量为14 697。结论建立的分离纯化方法易操作,成本低,得到的hLYZ纯度大于98%,该分离方法可用于hLYZ的规模化生产。
Objective To develop a method for isolation and purification of recombinant human lysozyme(hLYZ)and identify the purified product. Methods The hLYZ was isolated and purified by solid-liquid separation,membrane filtration,hydrophobic chromatography and ion exchange chromatography,and determined for purity by SDS-PAGE and HPLC,for potency according to the method in Chinese Pharmacopoeia(VolumeⅡ,2015 edition),for specificity by Western blot,and for relative molecular mass by matrix-assisted laser desorption/ionization time of flight mass spec-trometry(MALDITOF). Results Recombinant hLYZ with a single component was isolated by the developed method,of which the purity reached 99% by SDS-PAGE and 98. 8% by HPLC. Purified hLYZ showed specific binding to human anti-LYZ antibody,of which the activity was 1. 2 × 105 U/mg,and the relative molecular mass was 14 697. Conclusion The developed method for isolation and purification of h LYZ was easy to handle and of low cost. The purity of obtained hLYZ was more than 98%. The method may be used for the large-scale production of hLYZ.
Objective To develop a method for isolation and purification of recombinant human lysozyme(hLYZ)and identify the purified product. Methods The hLYZ was isolated and purified by solid-liquid separation,membrane filtration,hydrophobic chromatography and ion exchange chromatography,and determined for purity by SDS-PAGE and HPLC,for potency according to the method in Chinese Pharmacopoeia(VolumeⅡ,2015 edition),for specificity by Western blot,and for relative molecular mass by matrix-assisted laser desorption/ionization time of flight mass spec-trometry(MALDITOF). Results Recombinant hLYZ with a single component was isolated by the developed method,of which the purity reached 99% by SDS-PAGE and 98. 8% by HPLC. Purified hLYZ showed specific binding to human anti-LYZ antibody,of which the activity was 1. 2 × 105 U/mg,and the relative molecular mass was 14 697. Conclusion The developed method for isolation and purification of h LYZ was easy to handle and of low cost. The purity of obtained hLYZ was more than 98%. The method may be used for the large-scale production of hLYZ.
引文
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[17]WEN C W,ZHAO Y Q,SHI L,et al.Purification of lysozyme from egg white by combination of polyethylene glycol precipitation and aqueous two-phase extraction[J].Biotechnol Bull,2017,33(5):89-93.(in Chinese)闻崇炜,赵烨清,石莉,等.聚乙二醇沉淀联用双水相萃取法纯化蛋清溶菌酶的研究[J].生物技术通报,2017,33(5):89-93.
[18]左胜,吴佳欢,廖志勇.蛋清溶菌酶分离纯化研究进展[J].科协论坛,2013,(3):73-74.
[19]ZHAO L,CAI J Y,ZHOU L,et al.Efficient purification of lysozyme by one-step cation colum chromatography and its bacteriolytic activity assay[J].China Pharmacy,2013,24(1):30-32.(in Chinese)赵林,蔡金艳,周林,等.一步阳离子柱层析高效纯化溶菌酶及其溶菌活性[J].中国药房,2013,24(1):30-32.
[20]CHI X Y,GE Y H,HAN L Y,et al.Study on the extraction of lysozyme in egg-white[J].Hubei Agri Sci,2010,49(9):2212-2214.(in Chinese)迟晓艳,葛宜和,韩立亚,等.鸡蛋清中溶菌酶提取技术的研究[J].湖北农业科技,2010,49(9):2212-2214.
[2]SONG J.Separation methods and progress of lysozyme[J].Henan Chem Indust,2017,34(7):16-18.(in Chinese)宋娟.溶菌酶的分离方法及进展[J].河南化工,2017,34(7):16-18.
[3]HU X L,ZHOU G Y.Fresh-keeping effect of compound sodium alginate-lysozyme coating on Malu grapes[J].Food Sci,2011,32(20):271-276.(in Chinese)胡晓亮,周国燕.海藻酸钠和溶菌酶复合涂膜对马陆葡萄贮藏的保鲜效果[J].食品科学,2011,32(20):271-276.
[4]LIN L.Review on separation methods and development of lysozyme[J].Guangzhou Chem Indust,2012,40(7):53-56.(in Chinese)林立.溶菌酶分离方法综述及进展[J].广州化工,2012,40(7):53-56.
[5]APPENDINI P,HOTCHKISS J H.Immobilization of lysozyme on food contact polymers as potential antimicrobial films[J].Packag Technol Sci,1997,10(5):271-279.
[6]FU B,JI X L,YU H Y,et al.Preliminary study on isolation and extraction of lysoyme from egg white[J].Acta Agri Zhejiang,2013,25(6):1364-1367.(in Chinese)傅冰,季秀玲,俞汇颖,等.鸡蛋清中溶菌酶的分离提取[J].浙江农业学报,2013,25(6):1364-1367.
[7]FAN L L,LIN N,FENG X Q,et al.Research advances on lysozyme and its application in food industry[J].Food Ferment Indust,2015,41(3):248-252.(in Chinese)范林林,林楠,冯叙桥,等.溶菌酶及其在食品工业中的应用[J].食品与发酵工业,2015,41(3):248-252.
[8]YU H F.Study on highly efficient extraction and quantitative determination of lysozyme in chicken egg white[J].Wuhan:Huazhong Agricultural University,2010.(in Chinese)余海芬.蛋清中溶菌酶的高效提取及定量测定方法研究[D].湖北武汉:华中农业大学,2010.
[9]CHEN P,ZHAO W,LI L,et al.Adsorption characteristic of dextran affinity-ulttafittration escort to lysozyme[J].Sci Technol Food Indust,2011,32(8):84-86,89.(in Chinese)陈萍,赵玮,李琳,等.水溶性葡聚糖亲和-超滤载体对溶菌酶吸附分离特性的研究[J].食品工业科技,2011,32(8):84-86,89.
[10]DATTA D,BHATTACHAJEE S,NATH A,et al.Separation of ovalbumin from chicken egg white using two-stage ultrafiltration technique[J].Separat Purif Technol,2009,66(2):353-361.
[11]LAMPPA J W,TANYOS S A,GRISWOLD K E.Engineering Escherichia coli for soluble expression and single step purification of active human lysozyme[J].J Biotechnol,2013,164(1):1-8.
[12]WU S.Expression optimizing strategy of recombinant human lysozyme and the application of infrared Spectrometer on the substrate monitor in Pichia pastoris fermentation[D].Shanghai:East China University of Science and Technology,2014.(in Chinese)吴胜.毕赤酵母表达重组人溶菌酶的发酵工艺优化及傅里叶红外光谱仪在其过程底物控制中的初步应用研究[D].上海:华东理工大学,2014.
[13]LI Q Y.Application and development prospect of biological lysozyme[J].Mod Salt Chem Indust,2017,44(2):9-10.(in Chinese)李秋雁.生物溶菌酶的提取应用及发展前景研究[J].现代盐化工,2017,44(2):9-10.
[14]QI Y,SUN B,ZHAO X,et al.Establishment of extracting lysozyme from egg white in polyethylene glycol and dipotassium hydrogen phosphate aqueous two-phase system[J].China Poul,2013,35(5):22-26.(in Chinese)齐玉,孙波,赵晓,等.聚乙二醇/磷酸氢二钾双水相体系萃取蛋清溶菌酶方法的建立[J].中国家禽,2013,35(5):22-26.
[15]LIU Z M,ZHAO Z Y,LI J R,et al.Extracting lysozyme from egg white in polyethylene glycol sodium carbonate[J].The Guide of Sci Educat,2014,6(10):37-39.(in Chinese)刘志明,赵展英,李建荣,等.聚乙二醇/碳酸钠双水相萃取蛋清溶菌酶[J].科教导刊旬刊,2014,6(10):37-39.
[16]SHANGGUAN D C,HAN W J,SHANG Y F.Extraxting lysozyme from eggshell membrane in combination with polyethylene glycol-sodium carbonate aqueous two-phase system and resin[J].Biol Chem Engi,2016,2(3):43-44.(in Chinese)上官丁从,韩文进,商亚芳.聚乙二醇-碳酸钠双水相与树脂联用纯化蛋壳膜中的溶菌酶[J].生物化工,2016,2(3):43-44.
[17]WEN C W,ZHAO Y Q,SHI L,et al.Purification of lysozyme from egg white by combination of polyethylene glycol precipitation and aqueous two-phase extraction[J].Biotechnol Bull,2017,33(5):89-93.(in Chinese)闻崇炜,赵烨清,石莉,等.聚乙二醇沉淀联用双水相萃取法纯化蛋清溶菌酶的研究[J].生物技术通报,2017,33(5):89-93.
[18]左胜,吴佳欢,廖志勇.蛋清溶菌酶分离纯化研究进展[J].科协论坛,2013,(3):73-74.
[19]ZHAO L,CAI J Y,ZHOU L,et al.Efficient purification of lysozyme by one-step cation colum chromatography and its bacteriolytic activity assay[J].China Pharmacy,2013,24(1):30-32.(in Chinese)赵林,蔡金艳,周林,等.一步阳离子柱层析高效纯化溶菌酶及其溶菌活性[J].中国药房,2013,24(1):30-32.
[20]CHI X Y,GE Y H,HAN L Y,et al.Study on the extraction of lysozyme in egg-white[J].Hubei Agri Sci,2010,49(9):2212-2214.(in Chinese)迟晓艳,葛宜和,韩立亚,等.鸡蛋清中溶菌酶提取技术的研究[J].湖北农业科技,2010,49(9):2212-2214.