Two Different Proteases from Streptomyces hygroscopicus Are Involved in Transglutaminase Activation
Two Different Proteases from Streptomyces hygroscopicus Are Involved in Transglutaminase Activation
复制标题
吸水链霉菌的两种不同蛋白酶参与转谷氨酰胺酶激活
DOI：
10.1021/jf8008519
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发表时间：
2008-11-12
影响因子：
6.1
通讯作者：
Chen, Jian
中科院分区：
文献类型：
--
作者：
Zhang, Dongxu;Wang, Miao;Chen, Jian
转氨酶（TGase）是食品工业中唯一能够将共价键引入蛋白质的商业酶，在几种链霉菌中作为酶原（Pro-TGase）分泌。在以前的研究中，只有金属蛋白酶已被分离出茂原链霉菌作为一种内源性TGase激活蛋白酶（TAP）。在这项研究中，不仅内源性金属蛋白酶，而且内源性丝氨酸蛋白酶被发现参与TGase激活在吸水链霉菌。在无细胞系统中，TAP抑制剂首先用十六烷基三甲基溴化铵（CTAB）沉淀以保持TAP活性。随后，加入不同类型的蛋白酶抑制剂以鉴定参与S.吸湿性。TGase活化被1 mM苯甲磺酰氟（PIVISF）和10 mM乙二胺四乙酸（EDTA）抑制，这表明丝氨酸蛋白酶和金属蛋白酶参与TGase活化过程。此外，从S.吸湿性cus为丝氨酸蛋白酶，与S.茂原。此外，发现链霉菌Pro-TGases在TGase的N-末端之前具有保守的氨基酸序列，其含有丝氨酸蛋白酶和金属蛋白酶的切割位点。这些结果表明，内源性丝氨酸和金属磷酸酶都参与了S。吸湿性。据作者所知，这是第一个报告，内源性丝氨酸蛋白酶参与链霉菌TGase激活。
Transglutaminase (TGase), the only commercial enzyme in the food industry capable of introducing covalent bonds to proteins, is secreted as a zymogen (Pro-TGase) in several Streptomyces species. In previous studies, only a metalloprotease has been isolated from Streptomyces mobaraensis as an endogenous TGase-activating protease (TAP). In this study, not only an endogenous metalloprotease but also an endogenous serine protease is found to be involved in TGase activation in Streptomyces hygroscopicus. In a cell-free system, the TAP inhibitor was first precipitated with cetyltrimethyl ammonium bromide (CTAB) to maintain TAP activity. Subsequently, different types of protease inhibitors were added to identify the TAP involved in TGase activation in S. hygroscopicus. TGase activation was inhibited by 1 mM phenylmethanesulfonyl fluoride (PIVISF) and 10 mM ethylenediaminetetraacetic acid (EDTA), indicating the involvement of serine protease and metalloprotease in the TGase activation process. Furthermore, the TAP purified from a liquid culture of S. hygroscopi. cus was identified as a serine protease, which is different from the TAP isolated from S. mobaraensis. In addition, Streptomyces Pro-TGases were found to have a conserved amino acid sequence preceding the N-terminal of TGase, which contained cleavage sites for both serine protease and metalloprotease. These results indicate that endogenous serine and metal lop roteases are both involved in TGase activation in S. hygroscopicus. To the authors' knowledge, this is the first report that an endogenous serine protease is involved in Streptomyces TGase activation.