Bolumar Tomás, Sanz Yolanda, Aristoy M-Concepción, Toldrá Fidel
Instituto de Agroquímica y Tecnología de Alimentos (C.S.I.C.), Apartado 73, 46100 Burjassot, Valencia, Spain.
Int J Food Microbiol. 2003 Sep 1;86(1-2):141-51. doi: 10.1016/s0168-1605(03)00069-2.
A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides. The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.
从汉逊德巴利酵母CECT 12487中分离出一种由Co(2+)激活的金属精氨酰氨肽酶（EC 3.4.11.6）。该酶经硫酸鱼精蛋白沉淀后，依次通过弱阴离子交换色谱、凝胶过滤色谱和强阴离子交换色谱进行纯化。精氨酰氨肽酶（AAP）纯化了337倍，回收率为18%。AAP似乎是一种分子量为101 kDa的二聚体。该酶在pH值6至9的范围内具有活性。在pH 7.0和37℃时检测到最佳活性。AAP活性受到典型氨肽酶抑制剂（嘌呤霉素和抑氨肽酶素）、还原剂（二硫苏糖醇）、螯合剂（乙二胺四乙酸、乙二醇双乙醚二胺四乙酸和菲咯啉）以及巯基试剂（碘乙酸）的抑制。Ca(2+)、Mn(2+)和Co(2+)激活该酶，而Cu(2+)、Cd(2+)、Hg(2+)和Mg(2+)则抑制它。针对精氨酸-7-氨基-4-甲基香豆素（Arg-AMC）和亮氨酸-7-氨基-4-甲基香豆素（Leu-AMC）计算出的K(m)值分别为0.071和0.094 mM。该酶对碱性氨基酸（精氨酸和赖氨酸）表现出最大特异性，但也能够水解不带电荷的氨基酸（亮氨酸、甲硫氨酸和丙氨酸），并且对芳香族氨基酸（苯丙氨酸和酪氨酸）的水解速率较低。当酸性残基位于二肽的C末端位置时，AAP表现出更高的活性。所述从汉逊德巴利酵母中纯化精氨酰氨肽酶的方法有助于填补对香肠中最常分离出的酵母之一的外肽酶活性缺乏了解的空白，并有助于理解其在发酵香肠成熟过程中的作用。
