曲酸对南美白对虾酚氧化酶活性和结构的影响

Effect of Kojic Acid on Enzymatic Activity and Structure of Phenoloxidase from the Shrimp Penaeus vannamei

为研究曲酸对南美白对虾酚氧化酶(phenol oxidase,PO)活性和结构的影响,以南美白对虾头胸部分离纯化出的PO为原料,研究不同浓度曲酸(0、0.1、0.3、0.5、0.7、0.9、1.1、1.3、1.5 mmol/L)对PO活性的抑制,确定曲酸作用PO的抑制类型;通过测定不同浓度曲酸(0、0.1、0.3、0.5、0.7、0.9、1.1 mmol/L)处理的PO蛋白内源荧光光谱、表面疏水性和二级结构类型含量的变化,研究曲酸对PO空间结构的影响。结果表明:随着作用PO曲酸浓度的增加,PO的剩余酶活逐渐减少,当曲酸浓度达到1.5 mmol/L时,PO的剩余酶活仅为9.8%,参与的反应体系褐变指数变化值为3.02,抑褐变效果明显;曲酸能够有效的抑制PO活性,曲酸作用PO的抑制类型属于竞争性抑制剂;随着作用PO曲酸浓度的增加,PO的内源荧光强度不断降低,最大发射波长发生红移,PO蛋白表面疏水性先增加后降低;随着作用PO曲酸浓度的增加,二级结构类型α-螺旋和β-折叠含量降低,无规则卷曲和β-转角增加。可以推测,曲酸与PO结合后,酶蛋白空间结构发生了改变。

The effect of kojic acid on the enzymatic activity and structure of phenoloxidase(PO)from Penaeus vannamei was investigated.The PO enzyme purified from shrimp heads was treated with different concentrations(0-1.5mmol/L)of kojic acid.Kojic acid was found to be able to inhibit PO activity.The type of inhibition was determined and the effect of different concentrations(0-1.1mmol/L)of kojic acid on the spatial structure of the PO enzyme was examined by measuring changes in the intrinsic fluorescence spectrum,surface hydrophobicity and secondary structure contents.The results showed that the residual PO activity gradually decreased to9.8%of the initial activity with increasing kojic acid concentration up to1.5mmol/L,and the change in browning index was3.02,suggesting prominent browning inhibition.Kojic acidcould effectively inhibit PO activity,and the type of inhibition was competitive.The intrinsic fluorescence intensity of PO continuously decreased with increasing kojic acid concentration and the maximum emission wavelength(λmax)was red shifted.The surface hydrophobicity of PO decreased after an initial increase.In addition,as the concentration of kojic acid increased,theα-helix andβ-sheet contents decreased,butβ-turn and random coil increased.It was speculated that the spatial structure of PO was changed due to its reaction with kojic acid.