基于低温活性染料标记皮粉底物的蛋白酶性能表征方法
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摘要
建立了一种以低温活性染料标记的未变性皮粉(胶原纤维)为底物的快速测定蛋白酶活力的方法。优化了染色皮粉的制备方法,考察了底物的性质,优化了酶活力检测条件,并对几种蛋白酶的活力进行了测定。结果表明,染色后皮粉的结构未发生明显的改变,适合在较宽pH值范围进行蛋白酶活力测定,测定结果具有较好的重现性;蛋白酶不同底物作用性能有较大的差异,相对于酪蛋白和热变性染色皮粉,新建立的方法的测定结果对制革过程中酶的选用和使用条件的优化更具实际的指导意义。
A method for rapid determination of protease activity based on the substrate of un-denatured hide powder(collagen fibers) labeled with a low temperature reactive dyestuff was developed. The preparation method of the substrate was optimized, the properties of substrate were investigated, the conditions of detection were optimized, and the activities of several proteases were measured. The results show that the structure of the dyed hide powder does not change significantly, and it is suitable for the determination of protease activity in a wide pH range. The performances of proteases towards different substrates are different. Compared with casein and heat-denaturated staining powder, the results of the new method have more practical guiding significance for the selection of proteases and the optimization of using conditions in the process of leather making.
A method for rapid determination of protease activity based on the substrate of un-denatured hide powder(collagen fibers) labeled with a low temperature reactive dyestuff was developed. The preparation method of the substrate was optimized, the properties of substrate were investigated, the conditions of detection were optimized, and the activities of several proteases were measured. The results show that the structure of the dyed hide powder does not change significantly, and it is suitable for the determination of protease activity in a wide pH range. The performances of proteases towards different substrates are different. Compared with casein and heat-denaturated staining powder, the results of the new method have more practical guiding significance for the selection of proteases and the optimization of using conditions in the process of leather making.
引文
[1] Foroughi F,Keshavarz T,Evans C S.Specificities of proteases for use in leather manufacture[J].Journal of Chemical Technology&Biotechnology,2006,81(3):257-261.
[2] Edmonds R.Proteolytic de pilation of lambskins[D].New Zealand:Massey University,2008.
[3] Cupp-Enyard C.Sigma's Non-specific Protease Activity Assay-Casein as a Substrate[J].Journal of Visualized Experiments,2008(19).
[4] M S,G D,Bai.Hide power azure and azocoll as substrates for assay of the proteolytic activity of Bate[J].J Am Leather Chem As, 1992,(87):287-295.
[5] H R,Mc G,P S.A new ultrasensitive method for the determination of proteolytic activity[J]. Clin Chem Acta,1968,197-203(21).
[6]李成霞,许冰斌,王然然,等.以变性染色皮粉为底物的蛋白酶活力检测方法研究[J].中国皮革,2012,(05):19-23.
[7]孟春丽,曹机良,李建辉.L型活性染料对蚕丝的低温染色研究[J].丝绸,2014,(07):6-10.
[8]李倩,杨欢,李国英.[EMIM][AC]溶剂对Ⅰ型胶原结构的影响[J].皮革科学与工程,2017,(05):5-9.
[9]魏香奕,张文华,石碧.胶原重聚集纤维材料的制备和表征[J].功能材料,2014,(15):15143-15147.
[10]瞿怡,苏寒雨,刘文涛,等.超声波辅助法提取牛皮胶原蛋白[J].皮革科学与工程,2018,(02):5-9.
[11]刘萌,李建龙,但年华,等.牛肌腱胶原纤维的提取及其性能表征[J].中国皮革,2013,(01):19-22.
[12]李凤娟,林炜,程庆甦,等.新型胶原基pH敏感水凝胶的制备及性能[J].化工进展,2009,(03):441-444.
[2] Edmonds R.Proteolytic de pilation of lambskins[D].New Zealand:Massey University,2008.
[3] Cupp-Enyard C.Sigma's Non-specific Protease Activity Assay-Casein as a Substrate[J].Journal of Visualized Experiments,2008(19).
[4] M S,G D,Bai.Hide power azure and azocoll as substrates for assay of the proteolytic activity of Bate[J].J Am Leather Chem As, 1992,(87):287-295.
[5] H R,Mc G,P S.A new ultrasensitive method for the determination of proteolytic activity[J]. Clin Chem Acta,1968,197-203(21).
[6]李成霞,许冰斌,王然然,等.以变性染色皮粉为底物的蛋白酶活力检测方法研究[J].中国皮革,2012,(05):19-23.
[7]孟春丽,曹机良,李建辉.L型活性染料对蚕丝的低温染色研究[J].丝绸,2014,(07):6-10.
[8]李倩,杨欢,李国英.[EMIM][AC]溶剂对Ⅰ型胶原结构的影响[J].皮革科学与工程,2017,(05):5-9.
[9]魏香奕,张文华,石碧.胶原重聚集纤维材料的制备和表征[J].功能材料,2014,(15):15143-15147.
[10]瞿怡,苏寒雨,刘文涛,等.超声波辅助法提取牛皮胶原蛋白[J].皮革科学与工程,2018,(02):5-9.
[11]刘萌,李建龙,但年华,等.牛肌腱胶原纤维的提取及其性能表征[J].中国皮革,2013,(01):19-22.
[12]李凤娟,林炜,程庆甦,等.新型胶原基pH敏感水凝胶的制备及性能[J].化工进展,2009,(03):441-444.