A Novel<scp>L</scp>-Amino Acid Ligase from<i>Bacillus subtilis</i>NBRC3134, a Microorganism Producing Peptide-Antibiotic Rhizocticin
作者:Kuniki KINO、Yoichi KOTANAKA、Toshinobu ARAI、Makoto YAGASAKI
DOI:10.1271/bbb.80842
日期:2009.4.23
l-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected l-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel l-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess l-arginyl-l-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting l-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was l-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward l-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.
l-氨基酸连接酶以依赖 ATP 的方式催化未受保护的 l-氨基酸形成 α-肽键,这种酶对高效生产多肽非常有用。我们进行了酶纯化,从生产多肽抗生素根肿灵的微生物枯草芽孢杆菌(Bacillus subtilis NBRC3134)中获得了一种新型 l-氨基酸连接酶。根瘤菌素是一种二肽或三肽抗生素,通常含有 l-精氨酰-l-2-氨基-5-磷酰基-3-顺式戊烯酸。通过检测精氨酰羟氨酸盐合成活性进行纯化,最终纯化出目标酶 1280 倍,产率为 0.8%。然后克隆了相应的基因,并将其命名为 rizA,rizA 的长度为 1 242 bp,编码 413 个氨基酸残基。研究人员制备了重组 RizA,发现重组 RizA 能以 ATP 依赖性方式合成 N 端为 l-精氨酸的二肽。RizA对N端底物精氨酸具有严格的底物特异性,而对C端的底物特异性则比较宽松。