青鳞鱼肌肉蛋白复合酶水解机制研究

Mechanism Study on the Enzymolysis of Muscle Proteins from Harengula Zunasi

本文研究了海水鱼青鳞鱼(Harengula Zunasi Bleeker)肌肉蛋白由木瓜蛋白酶和风味酶复合而成的复合酶水解机制。当水解时间固定为120min时,采用正交试验法优选得到复合酶水解的适宜条件为酶浓度为7.5×103IU/g蛋白质、温度为45℃、pH6.5和底物浓度为5.5g蛋白质%。在此适宜的水解条件下,蛋白质水解度与水解时间的关系曲线类似于已公开发表的沙丁鱼、小龙虾、鲱鱼及鲑鱼的酶水解曲线;在30～210min的水解时间内,水解机理可以用三个线性方程式来描述:DH%=0.026×水解时间+8.85 (r=0.98)、产物游离氨基酸总量TFAA =0.16×水解时间+38.76 (r=0.99)和产物平均链长MLPL=-0.019×水解时间+10.79 (r=-0.96)。产物游离氨基酸分析结果揭示了复合酶对胱氨酸、苯丙氨酸和酪氨酸结合的肽键有最高特异性,而对色氨酸、脯氨酸、天门冬氨酸、丝氨酸、甘氨酸和谷氨酸有最低特异性; 同时,在210min之前达到最大水解力(HC)的游离氨基酸或多或少又参与了新的肽键的形成,揭示复合酶对青鳞鱼肌肉蛋白质不仅具有水解作用而且还有合成作用。水解产物含氮量分析表明复合酶水解产物经离心、0.45 μm和0.22 μm微孔过滤所得到的三种类型的水解产物的含氮化合物在分子大小方面有一定的差异。

Mechanism of the enzymolysis of muscle proteins from Harengula Zunasi Bleeker by compound enzyme that obtainedfrom mixture with papain and flavorease was studied. The optimum conditions of compound enzyme for enzymolysis muscle proteinare at enzyme consistency 7.5×103IU/g protein, temperature 45℃, pH6.5 and substrate consistency 5.5g protein % throughorthogonal trial with the constant time of 2 h. Under the optimum conditions, there are 3 linear relationship models expressed asDH%=0.026×[hydrolysis time]+8.85 (r=0.98), TFAA (total free amino acids of hydrolysate) =0.16×[hydrolysis time]+38.76(r=0.99) and MLPL(mean length of peptide linkage of hydrolysate)=-0.019×[hydrolysis time]+10.79(r=-0.96).The analysisof free amino acids revealed that compound enzyme has the strongest hydrolysis specificity toward the peptide linkages suppliedby cysteine and phenylalanine+tyrosine and the lowest hydrolysis specificity toward the peptide bonds supplied by tryptophane,proline, asparaginic acid, serine, glycine and glutamic acid. Meanwhile, the amino acids arriving at maximum hydrolyzing capac-ity before 210 min may have participated in the forming of the new peptide bonds at certain extent after arriving at themselvesmaximum hydrolyzing capacity. The determining of nitrogen content revealed that there is significant difference of the moleculesize of nitrogenous compositions among centrifugal hydrolysates, 0.45 μm hydrolysates and 0.22 μm hydrolysates at sametime of a range from 30 to 210 min because their nitrogen content has significant difference. The determining of molecularweight revealed that there is no significant difference in mean of relative molecular weight of all centrifugal hydrolasates and therelative molecular weight being from 300 to 1200 is covered 66% to 78% of total of the molecule number in centrifugal hydroly-sates. It is possible to give fundament and recommendations for the all-around utilization of Harengula Zunasi due to this work.