摘要
目的:探究鲢鱼、青鱼内源性转谷氨酰胺酶(transglutaminase,TGase)的酶学性质差异。方法:采用80%(NH_(4))_(2)SO_(4)盐析、Q-Sepharose FF和Sephacryl S-200 HR层析法从鲢鱼、青鱼肌肉中分离纯化出鲢鱼TGase(STG)、青鱼TGase(BTG),并对酶的相对分子质量、肽段序列、二级结构、适宜反应条件、热失活动力学等指标进行测定。结果:纯化后的STG和BTG的比酶活分别为14.34,12.67 U/mg,二者具有相近的相对分子质量。二者的肽段序列存在一定差异,其二级结构均以β-折叠为主,但STG的β-折叠含量略高于BTG的。STG和BTG的适宜反应温度均为50℃,适宜反应pH分别为8.0,7.5,完全激活两种TGase活性所需的Ca^(2+)浓度均为1 mmol/L,DTT可使两种TGase的酶活性增强,而PMSF、NH_(4)Cl、NEM、EDTA、Cu^(2+)、Ba^(2+)、Zn^(2+)、Mg^(2+)则会抑制其酶活。当温度为37~50℃时,热处理对STG和BTG的钝化均符合一级指数衰减动力学,二者的动力学参数E a值相近。结论:STG和BTG的一级结构及二级结构差异明显,但仍具有相似的适宜反应条件和热失活动力学特征。
Objective:This study aimed to investigate the differences in enzymatic properties of endogenous transglutaminase(TGase)in silver carp and black carp.Methods:STG and BTG were purified from the muscle of silver carp and black carp,respectively,by 80%ammonium sulfate precipitation,Q-Sepharose FF,and Sephacryl S-200 HR chromatographies.Two enzymes were analyzed for relative molecular weights,peptide sequences,secondary structures,optimal reaction conditions,and thermal inactivation kinetics.Results:The purified STG and BTG showed similar relative molecular weights,of which the enzyme activities were 14.34 U/mg and 12.67 U/mg,respectively.Both enzymes showed differences in peptide sequences.The secondary structures of them were mainly theβ-fold,though the content ofβ-fold in STG was slightly higher than that of BTG.The optimal temperatures for STG and BTG were both 50℃,and the optimal pH values were 8.0 and 7.5,respectively.The enzymes required Ca^(2+)up to 1 mmol/L for full activation.The activities of STG and BTG were enhanced by DTT,whereas PMSF,NH_(4)Cl,NEM,EDTA,Cu^(2+),Ba^(2+),Zn^(2+),and Mg^(2+)showed inhibitory effects.When the temperature was 37~50℃,the passivations of STG and BTG by thermal treatment conformed to the first-order exponential decay kinetics with similar values of E a.Conclusion:The primary and secondary structures of STG and BTG exhibited obvious differences,yet they still exhibited similar properties in terms of optimal reaction conditions and thermal inactivation kinetics.
作者
易林
安玥琦
刘茹
胡杨
熊善柏
YI Lin;AN Yueqi;LIU Ru;HU Yang;XIONG Shanba(College of Food Science and Technology,Huazhong Agricultural University,Wuhan,Hubei 430070,China;National R&D Branch Center for Conventional Freshwater Fish Processing〔Wuhan〕,Huazhong Agricultural University,Wuhan,Hubei 430070,China)
出处
《食品与机械》
CSCD
北大核心
2023年第10期4-12,共9页
Food and Machinery
基金
财政部和农业农村部:国家现代产业技术体系(编号:CARS-45-28)。
关键词
淡水鱼
转谷氨酰胺酶
分离纯化
结构特性
适宜反应条件
失活动力学
freshwater fish
transglutaminase
purification
structure properties
optimal reaction conditions
inactivation kinetics