Purification, Characterization, Molecular Cloning, and Expression of a New Aminoacylase from<i>Streptomyces mobaraensis</i>That Can Hydrolyze<i>N</i>-(Middle/Long)-chain-fatty-acyl-<scp>L</scp>-amino Acids as Well as<i>N</i>-Short-chain-acyl-<scp>L</scp>-amino acids
作者:Mayuko KOREISHI、Yasuyuki NAKATANI、Manami OOI、Hiroyuki IMANAKA、Koreyoshi IMAMURA、Kazuhiro NAKANISHI
DOI:10.1271/bbb.90081
日期:2009.9.23
We report here on the purification, characterization, molecular cloning, and expression of a new aminoacylase, initially isolated from the supernatant of Streptomyces mobaraensis (Sm-AA). Purified wild-type Sm-AA was found to be a monomeric protein with a molecular mass of 55 kDa. The cloned gene of Sm-AA contained an ORF of 1,383 bp, encoding a polypeptide of 460 amino acids. A BLAST search revealed that Sm-AA belongs to the peptidase M20 family, with identities to a hypothetical protein from Streptomyces pristinaespiralis, a putative peptidase from Streptomyces avermitilis, peptidase M20 from Frankia sp., succinyl-diaminopimelate desuccinylase from Hemophilus influenzae, and aminoacylase-1 from porcine kidney at 89, 88, 67, 29, and 25% respectively. The Sm-AA gene was subcloned into an expression vector, pSH19, and was expressed in Streptomyces lividans TK24. The amount of the recombinant Sm-AA expressed in the S. lividans cells was approximately 42-fold higher than that of Sm-AA found in the supernatant of S. mobaraensis. Sm-AA showed high hydrolytic activity towards various N-acetyl-l-amino acids and N-(middle/long)-chain-fatty-acyl-l-amino acids, with a preference for the acyl derivatives of l-Met, l-Ala, l-Cys, etc. with an optimum pH and temperature for reaction of about 7.5 and 50 °C (at pH 7.5).
我们在此报告了一种新的氨酰酶的纯化、特征化、分子克隆和表达,该酶最初从放线菌Mobaraensis(Sm-AA)的上清液中分离出来。纯化的野生型Sm-AA被发现是单体蛋白,分子量为55 kDa。克隆的Sm-AA基因包含一个1,383 bp的开放阅读框(ORF),编码460个氨基酸的多肽。BLAST搜索显示,Sm-AA属于肽酶M20家族,与来自Streptomyces pristinaespiralis的假设蛋白、来自Streptomyces avermitilis的假定肽酶、来自Frankia sp.的肽酶M20、来自流感嗜血杆菌的琥珀酰二氨基庚酸去琥珀酰酶,以及来自猪肾的氨酰酶-1的同源性分别为89%、88%、67%、29%和25%。Sm-AA基因被亚克隆入表达载体pSH19,并在Streptomyces lividans TK24中表达。重组Sm-AA在S. lividans细胞中的表达量约为S. mobaraensis上清液中Sm-AA的42倍。Sm-AA对各种N-乙酰-l-氨基酸和N-(中/长)-链脂肪酰-l-氨基酸表现出高水解活性,特别偏好l-Met、l-Ala、l-Cys等的酰基衍生物,其最佳反应pH和温度约为7.5和50°C(在pH 7.5下)。