基于点击化学的蛋白酶修饰及其应用
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摘要
以葡聚糖为修饰剂,通过点击化学反应修饰蛋白酶(Savinase),增大蛋白酶分子量,从而更好地应用于羊毛生物法防毡缩加工。通过SDS-PAGE凝胶电泳、圆二色谱法和荧光光谱法考察蛋白酶修饰前后的结构变化,并测定其酶学性质和酶促动力学参数。结果表明,修饰后蛋白酶分子量明显增大,二级结构有所变化,酶与底物亲和力略有降低,但温度和p H值对酶活的影响情况相似。在羊毛防毡缩整理加工中,修饰蛋白酶处理的羊毛织物毡缩率满足"机可洗"标准,但强力损失明显减小。
Savinase was modified with dextran via click chemistry so as to enlarge the molecular weight,which can be applied for shrink resistance of wool. The structure changes of modified Savinase were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE),circular dichroism( CD) and fluorescence spectroscopy. Enzymatic properties and kinetic parameters were also determined. The results indicated that the molecular weight of the modified Savinase was significantly increased. After modification,the secondary structure of enzyme changed and the substrate affinity lowered,while the effects of temperature and p H on enzyme activities were similar. For the shrink-resistance finishing of wool,it could be found that the wool fabric treated by modified Savinase could meet the machine-washable standard with obviously decreased strength loss.
Savinase was modified with dextran via click chemistry so as to enlarge the molecular weight,which can be applied for shrink resistance of wool. The structure changes of modified Savinase were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis( SDS-PAGE),circular dichroism( CD) and fluorescence spectroscopy. Enzymatic properties and kinetic parameters were also determined. The results indicated that the molecular weight of the modified Savinase was significantly increased. After modification,the secondary structure of enzyme changed and the substrate affinity lowered,while the effects of temperature and p H on enzyme activities were similar. For the shrink-resistance finishing of wool,it could be found that the wool fabric treated by modified Savinase could meet the machine-washable standard with obviously decreased strength loss.
引文
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[20]王玉琪,巫光宏,林先丰,等.多糖和糖蛋白聚丙烯酰胺凝胶电泳染色方法的改进[J].植物生理学通讯,2009,45(2):169-172.
[21]余雪满,李清政.双氧水前处理对毛织物防毡缩整理的影响[J].印染助剂,2017,34(12):39-41,45.
[22]陈忍忍.蛋白酶的化学修饰及其在羊毛防毡缩整理中的应用[D].无锡:江南大学,2012.
[23]吴明和.圆二色光谱在蛋白质结构研究中的应用[J].氨基酸和生物资源,2010,32(4):77-80.
[24]RANJBAR B,GILL P. Circular dichroism techniques:biomolecular and nanostructural analyses-a review[J]. Chemical Biology&Drug Design,2009,74(2):101-120.
[2]黄庞慧,王强,董爱学,等.角质酶/角蛋白酶一浴法处理对羊毛性能的影响[J].生物学杂志,2014,31(6):94-98,102.
[3]范雪荣,黄庞惠,王强.羊毛的酶法防毡缩整理综述[J].针织工业,2015,3:30-35.
[4]SCHROEDER E,ZHANG Q,SHEN J,et al. Modification of esperase by covalent bonding to Eudragit polymers L 100 and S 100 for wool fiber surface treatment[J]. Biocatalysis and Biotransformation,2008,26(5):391-398.
[5]SMITH E,SCHROEDER M,GUEBITZ G,et al. Covalent bonding of protease to different sized enteric polymers and their potential use in wool processing[J]. Enzyme and Microbial Technology, 2010,47(3):105-111.
[6]SCHROEDER E,SCHWEITZER M,LENTING H B M,et al. Chemical modification of proteases for wool cuticle scale removal[J]. Biocatalysis and Biotransformation,2004,22(5/6):299-305.
[7]SHEN J,RUSHFORTH M,CAVACO-PAULO A,et al. Development and industrialization of enzymatic shrink-resist process based on modified proteases for wool machine washability[J]. Enzyme and Microbial Technology,2007,40(7):1656-1661.
[8]SILVA C J S M,PRABAHARAN M,GUBITZ G,et al. Treatment of wool fibers with subtilisin and subtilisin-PEG[J]. Enzyme and Microbial Technology,2005,36(7):917-922.
[9]ABDEL-NABY M A,FOUAD A A,EL-REFAI H A. Catalytic and thermodynamic properties of glycosylated Bacillus cereus cyclodextrin glycosyltransferase[J]. International Journal of Biological Macromolecules,2015,76:132-137.
[10]杨淑凤,邓国英,刘欣,等.钙粘蛋白糖基化修饰的研究进展[J].生物学杂志,2016,33(2):100-102.
[11]申璇,袁久刚,王强,等.糖化改性对胶原蛋白乳化性能的影响[J].生物学杂志,2017,34(5):20-23,42.
[12]姜炎甫,邹树平,轩秀玲,等.酶的化学糖基化修饰的研究进展[J].精细与专用化学品,2017,25(6):1-6.
[13]KOLB H C,FINN M G,SHARPLESS K B. Click chemistry:diverse chemical function from a few good reactions[J]. Angewandte Chemie International Edition,2001,40(11):2004-2021.
[14]PALOMO J M. Click reactions in protein chemistry:from the preparation of semisynthetic enzymes to new click enzymes[J]. Organic and Biomolecular Chemistry,2012,10(47):9309-9318.
[15]杨帆,杨小雁,孔英俊,等.基于点击化学的琼脂糖微球上溶菌酶定点偶联方法研究[J].生物学杂志,2016,33(1):104-108.
[16]GODDARD-BORGER E D,STICK R V. An efficient,inexpensive,and shelf-stable diazotransfer reagent:imidazole-1-sulfonyl azide hydrochloride[J]. Organic Letters,2007,9(19):3797-3800.
[17]VAN DONGEN S F M,TEEUWEN R L M,NALLANI M,et al. Single-step azide introduction in proteins via an aqueous diazo transfer[J]. Bioconjugate Chemistry,2009,20(1):20-23.
[18]HELLER D A,LEVI L Y,PELET J M,et al. Modular‘click-in-emulsion’bone-targeted nanogels[J]. Advanced Materials,2013,25(10):1449-1454.
[19]李晓媛.“点击化学”制备牛血清白蛋白-聚乙二醇键合体系及其用于5-氟尿嘧啶的载药研究[D].长春:吉林大学,2009.
[20]王玉琪,巫光宏,林先丰,等.多糖和糖蛋白聚丙烯酰胺凝胶电泳染色方法的改进[J].植物生理学通讯,2009,45(2):169-172.
[21]余雪满,李清政.双氧水前处理对毛织物防毡缩整理的影响[J].印染助剂,2017,34(12):39-41,45.
[22]陈忍忍.蛋白酶的化学修饰及其在羊毛防毡缩整理中的应用[D].无锡:江南大学,2012.
[23]吴明和.圆二色光谱在蛋白质结构研究中的应用[J].氨基酸和生物资源,2010,32(4):77-80.
[24]RANJBAR B,GILL P. Circular dichroism techniques:biomolecular and nanostructural analyses-a review[J]. Chemical Biology&Drug Design,2009,74(2):101-120.
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