鲢内源性转谷氨酰胺酶的纯化及其性质

Purification and some enzymatic properties of transglutaminase from silver carp

以鲢(Hypophthalmichthys molitrix)肌肉为原料,采用硫酸铵盐析、凝胶过滤层析(Sephacryl S-300HR)和阴离子交换层析(DEAE-Sepharose FF)对鲢内源性转谷氨酰胺酶(TGase)分离纯化,并对其酶学性质进行研究。结果表明,酶液经多步纯化后可得单一酶蛋白,其分子质量为100ku,比活力为126.7U/mg,纯度提高34.2倍,该酶适宜的反应pH和温度分别为8.0和50℃。鲢内源性TGase具有Ca^(2+)依赖性,最适Ca^(2+)浓度为3mmol/L。DTT在低浓度(0~5 mmol/L)范围能增加酶的活性,而高浓度的DTT、EDTA及Mg^(2+)、Cu^(2+)、Zn^(2+)、Ba^(2+)、Sn^(2+)、Fe^(3+)等金属离子则表现出抑制作用。鲢内源性TGase对鲢和草鱼肌球蛋白有较强的催化交联作用,能够诱导鲢、草鱼肌球蛋白交联形成多聚体,且对鲢肌球蛋白的催化交联作用更强。

Transglutaminase(TGase)from silver carp was purified with ammonium sulfate precipitation,gel filtration on Sephacryl S-300 high resolution and DEAE-Sepharose fast flow ion-exchange chromatography.Some kinetic properties of transglutaminase purified were studied.The results showed that the molecular mass of the purified enzyme was 100 ku.The enzyme exhibited final purification fold of 34.2,specific activity of 126.7 U/mg.Results of further assaying enzymatic characterization showed that the purified enzyme had maximal activity at 30℃and pH 7.5.The activity of transglutaminase was slightly activated by Ca 2+and dithiothreitol(DTT)and strongly inhibited by ethylenediaminetetraacetic acid(EDTA).The metal ions Mg 2+,Cu 2+,Zn 2+,Ba 2+,Sn 2+,Fe 3+strongly inhibited transglutaminase activity as well.The transglutaminase can catalyze covalent cross-linking of mysion heavy chain(MHC)from silver carp and grass carp.The effects of the transglutaminase on mysion from silver carp were more noticeable than that from grass carp.