吸水链霉菌谷氨酰胺转氨酶两种同源异形体的鉴定及性质分析

Identification and properties of two isoforms of Streptomyces hygroscopicus transglutaminase

目的探讨吸水链霉菌谷氨酰胺转氨酶(transglutaminase,TGase,EC 2.3.2.13)的两种同源异形体成熟酶TGases A和TGases B的分子差异,并进行系统分析和鉴定。方法吸水链霉菌培养上清液经阴离子交换层析和凝胶过滤层析分离纯化后,将纯化产物再经阴离子交换层析,分析纯化产物的分子表面带电性质,通过基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)和远紫外圆二色谱(far ultra-violet circular dichroism spectrometry,Far-UV CD)法分析纯化产物的肽段序列及二级结构的差异,并检测成熟酶N-末端氨基酸序列。结果经阴离子交换层析和凝胶过滤层析分离纯化获得TGases的两种成熟酶TGases A和TGases B,均具有酶活性;TGase A和TGase B成熟酶分子具有不同的表面带电性质,酶活分别为(28.33±1.45)U/mg和(22.54±1.77)U/mg;TGases A和TGases B在质荷比(m/z)4 536.481和4 873.393处两个低丰度肽段有差异,但N-末端6个氨基酸序列完全相同;两者二级结构均以α-螺旋为主,但二级结构存在一定差异。结论吸水链霉菌TGases A和TGases B是由同一种酶原活化产生的同源异形体,两者的表面电荷性质、肽段序列及分子二级结构均有差异。

Objective To investigate the molecular differences between two isoforms（TGase A and TGase B） of Streptomyces hygroscopicus transglutaminase（TGase,EC2.3.2.13）,and analyze their properties.Methods The culture supernatant of S.hygroscopicus was purified by anion exchange chromatography and gel-filtration chromatography,and the purified TGase A and TGase B were analyzed for surface electric charge properties by anion exchange chromatography,and for differences in peptide sequences and secondary structures by matrix-assisted laser-desorption / ionization time of flight mass spectrometry（MALDI-TOF MS） and far-ultraviolet circular dichroism spectroscopy（Far-UV CD）.The amino acids at N-terminuses of TGase A and TGase B were sequenced.Results TGase A and TGase B with enzyme activities were obtained by anion exchange chromatography and gel filtration chromatography.Mature TGase A and TGase B molecules showed different surface electric charge properties,of which the enzyme activities were（28.33 ± 1.45） and（22.54 ± 1.77） U / mg respectively.TGase A and TGase B showed differences in two peptides with low abundances at m / z ratios of 4 536.481 and 4 873.393 respectively,of which the sequences of six amino acids at N-terminus were in agreement.Both the secondary structures of the two isoforms were mainly α-helices,though a certain difference was observed.Conclusion TGase A and TGase B were isoforms derived by activation of the same zymogen,of which the surface electric charge properties,peptide sequences and secondary structures showed certain differences.