Cloning, Purification, and Characterization of Tripeptidyl Peptidase from Streptomyces herbaricolor TY-21
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Cloning, Purification, and Characterization of Tripeptidyl Peptidase from Streptomyces herbaricolor TY-21 Keisuke Ekino 1 & Shinichi Yonei 1 & Hiroshi Oyama 2 & Takuji Oka 1 & Yoshiyuki Nomura 1 & Takashi Shin 1
Received: 1 May 2017 / Accepted: 21 June 2017 # Springer Science+Business Media, LLC 2017
Abstract Tripeptidyl peptidase (TPP) is an exopeptidase that sequentially hydrolyzes tripeptides from the N-terminus of oligopeptides or polypeptides. We performed screening for isolating novel TPP-producing microorganisms from soil samples. TPP activity was observed in the culture supernatant of Streptomyces herbaricolor TY-21 by using Ala-AlaPhe-p-nitroanilide (pNA) as the substrate. TPP from the culture supernatant was purified to approximately 790-fold. It was shown to cleave oxidized insulin B-chain, thereby with releasing tripeptide units, but not the N-terminal-protected peptide, Cbz-Ala-Ala-Phe-pNA. The TPP gene, designated tpp, was isolated from a partial genomic DNA library of S. herbaricolor TY-21. The TPP gene consisted of 1488 bp, and encoded a 133-amino acid pre-pro-peptide and a 362-amino acid mature enzyme containing conserved amino acid residues (Asp-36, His-77, and Ser-282) similar to the catalytic residues in subtilisin. TY-21 TPP belonged to the peptidase S8A family in the MEROPS database. The mature TY-21 TPP showed approximately 49% identity with tripeptidyl peptidase subtilisin-like (TPP S) from Streptomyces lividans strain 66. Keywords Tripeptidyl peptidase . Streptomyces herbaricolor . Subtilisin . Tripeptide . Peptidase S8A
* Keisuke Ekino [email protected]
1
Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan
2
Department of Life Science, Faculty of Science and Engineering, Setsunan University, Neyagawa, Osaka, Japan
Appl Biochem Biotechnol
Introduction Tripeptidyl peptidases (TPPs) are unique exopeptidases that cleave tripeptide units from oligopeptides or polypeptides. It is required to degrade unnecessary proteins in cells for sustaining life. In this process, TPP plays an important role and is present in lysosomes and the cytosol [1]. There are two kinds of TPPs in mammals [2], TPPI (EC 3.4.14.9) and TPPII (EC 3.4.14.10). TPPI has been isolated and purified from different sources such as the pituitary gland [3], pig ovary [4], and human osteoclastoma cells [5]. It is located in the lysosomes with an optimum pH of 4.0– 5.0. TPPII, which is widely distributed in the eukaryotic cells, is a high molecular mass peptidase, and shows optimum activity at neutral pH. It has been isolated from human erythrocytes [6], rat brain [7], and Drosophila melanogaster [8]. Additionally, TPP from microorganisms such as Streptomyces lividans 66 [9–12], Aspergillus fumigatus [13], and Streptomyces mobaraensis [14, 15] have been characterized. S. lividans is capable of secreting expressed proteins in the culture medium; therefore, it is used as a host for the production of numerous heterologous proteins [16, 17]. However, it was revealed that the he
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