黄粉虫凝乳蛋白酶的分离纯化及结构预测

Purification and structure prediction of milk-clotting protease from Tenebrio molitor larvae

黄粉虫资源丰富、蛋白含量高,具有很高的开发价值,然而关于黄粉虫凝乳蛋白酶鲜有报道。该研究采用连续柱层析的方法从黄粉虫肠道中分离纯化出了一种凝乳蛋白酶。利用液相色谱串联质谱和Edman降解等方法比对得到该酶的氨基酸序列。使用生物信息学方法预测和分析了其理化性质和结构。结果表明,黄粉虫凝乳蛋白酶的分子质量约为29.68 kDa,凝乳活力为173.8 SU/mg,C/P(clotting activity/proteolytic activity)值为104.7。该酶的等电点为4.22;疏水性氨基酸比例为43.37%,不稳定性指数31.49,理化性质稳定。与小牛皱胃酶和骆驼凝乳酶比对发现,三者在N端序列和二级结构等方面具有较大差异;该酶64.8%的氨基酸残基的归一化B因子(normalized B-factor,BFP)值<0,说明其三级结构具有良好的稳定性。该研究可为昆虫凝乳蛋白酶的研究提供理论依据。

Tenebrio molitor larvae,as an abundant resource,has profuse protein content,and good application potential,however,there are few reports on milk-clotting protease from T.molitor larvae.This study purified a milk-clotting protease from guts in T.molitor larvae by column chromatography.By liquid chromatography tandem mass spectrometry and Edman degradation,amino acid sequence of the protease was obtained.And the physicochemical properties and structure were predicted and analyzed based on bioinformatics.The results showed that a milk-clotting protease with a molecular mass of 29.68 kDa and a milk-clotting activity of 173.8 SU/mg was obtained.The protease had a C/P ration of 104.7,an isoelectric point of 4.22,a hydrophobic amino acid of 43.37%and an instability index of 31.49,indicating that its physicochemical properties were stable.Compared with bovine chymosin and camel chymosin,their N-terminal sequence and secondary structure had great differences.Since the normalized B-factor value of 64.8%amino acid residues were negative,this protease had a good stability.This study provides a theoretical basis for the research of insect-derived milk-clotting protease.