Gene cloning and overproduction of an aminopeptidase from Streptomyces septatus TH-2, and comparison with a calcium-activated enzyme from Streptomyces griseus

Biochemical and Biophysical Research Communications
Jiro ArimaTadashi Hatanaka

Abstract

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 pKas 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.

References

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Citations

Nov 4, 2006·Applied Microbiology and Biotechnology·Tadashi HatanakaMasaki Iwabuchi
Dec 25, 2012·World Journal of Microbiology & Biotechnology·Jiro ArimaNobuhiro Mori
Feb 4, 2009·Applied Biochemistry and Biotechnology·Bo WuBin Yao
Oct 13, 2010·Applied Biochemistry and Biotechnology·Arya S NandanKesavan Madhavan Nampoothiri
Jan 15, 2014·Enzyme and Microbial Technology·Arya S Nandan, Kesavan Madhavan Nampoothiri
Sep 25, 2007·Journal of Applied Microbiology·V NagyG Szakacs
Feb 22, 2013·Journal of the Science of Food and Agriculture·Xinxing GaoZhemin Zhou
Jan 11, 2005·Archives of Biochemistry and Biophysics·Tadashi HatanakaMasaki Iwabuchi
Jul 30, 2011·Metallomics : Integrated Biometal Science·Lee Macomber, Robert P Hausinger
Jul 17, 2018·Applied Biochemistry and Biotechnology·Kun WanTadashi Hatanaka
Apr 29, 2020·Applied Microbiology and Biotechnology·Arya Nandan, Kesavan Madhavan Nampoothiri
Dec 2, 2020·International Journal of Biological Macromolecules·Qin QinZhengfei Yan

Related Concepts

Aminopeptidase
Calcium
Enzyme Activation
Enzyme Stability
Hydrogen-Ion Concentration
Proteins, Recombinant DNA
Streptomyces
Streptomyces griseus
Homologous Sequences, Amino Acid

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