Gene cloning and overproduction of an aminopeptidase from Streptomyces septatus TH-2, and comparison with a calcium-activated enzyme from Streptomyces griseus
Gene cloning and overproduction of an aminopeptidase from Streptomyces septatus TH-2, and comparison with a calcium-activated enzyme from Streptomyces griseus
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DOI：
10.1016/j.bbrc.2004.03.082
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发表时间：
2004-04-30
影响因子：
3.1
通讯作者：
Hatanaka, T
中科院分区：
文献类型：
--
作者：
Arima, J;Iwabuchi, M;Hatanaka, T
从链霉菌TH-2（StreptomycesseptatusTH-2）中分离得到一种热稳定的氨肽酶（SSAP），并对其基因进行了克隆和测序。SSAP的一级结构与灰色链霉菌氨基肽酶（SGAP）的一级结构有71%的同源性，但缺少一个独特的钙结合位点。重组SSAP在大肠杆菌pET-KmS 2的培养上清中得到高效表达。重组SSAP和SGAP的比较表明，这两种酶是不同的钙和底物特异性的调制。SSAP的活性不受钙的调节，而SGAP是一种钙激活酶。SSAP有效地催化L-Lys-pNA的水解，而SGAP的L-Lys-pNA水解的反应速率显著低。此外，在SGAP中，Ca ~（2+）的存在降低了L-Lys-pNA水解的反应速率。SSAP也有不同的pK（a）s的反应，从SGAP，虽然几乎所有的残基组成的活性位点是保守的，在这两种酶。这一结果表明，SSAP具有不同于SGAP的底物结合环境和活性位点。(C)2004年爱思唯尔公司All rights reserved.
An aminopeptidase secreted from Streptomyces septatus TH-2 (SSAP) was identified as a heat stable enzyme, and the Ssap gene was cloned and sequenced. The primary structure of SSAP showed 71% identity with that of a Streptomyces griseus aminopeptidase (SGAP), however, it lacked a unique calcium binding site. The recombinant SSAP was overexpressed in the culture supernatant of Escherichia coli harboring pET-KmS2. A comparison of recombinant SSAP and SGAP showed that both enzymes are different in terms of modulation by calcium and substrate specificity. The activity of SSAP was not modulated by calcium, while SGAP is a calcium-activated enzyme. SSAP catalyzed the hydrolysis of L-Lys-pNA efficiently whereas the reaction rate for L-Lys-pNA hydrolysis of SGAP was significantly low. Furthermore, in SGAP, the presence of Ca2+ decreased the reaction rate Of L-Lys-pNA hydrolysis. SSAP also had different pK(a)s of reaction from that of SGAP, although almost all the residues which compose the active site were conserved in both enzymes. This result indicates that SSAP has a different environment of substrate binding and active sites from those of SGAP. (C) 2004 Elsevier Inc. All rights reserved.