title: Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.
abstract: A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes.
authors: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade Universitária, 50670-420, Recife, PE, Brazil.
authors: Renata Cristina da Penha
authors: RCDP
authors: França
authors: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade Universitária, 50670-420, Recife, PE, Brazil.
authors: Caio Rodrigo Dias
authors: CRD
authors: Assis
authors: Universidade Federal Rural de Pernambuco, Unidade Acadêmica de Serra Talhada, Av. Gregório Ferraz Nogueira, S/N - José Tomé de Souza Ramos, Serra Talhada, PE, CEP 56909-535, Brazil.
authors: Juliana Ferreira
authors: JF
authors: Santos
authors: Departamento de Bioquímica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors: Ricardo José Soares
authors: RJS
authors: Torquato
authors: Departamento de Bioquímica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors: Aparecida Sadae
authors: AS
authors: Tanaka
authors: Departamento de Biofísica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors: Izaura Yoshico
authors: IY
authors: Hirata
authors: Departamento de Biofísica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors: Diego Magno
authors: DM
authors: Assis
authors: Departamento de Biofísica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors: Maria Aparecida
authors: MA
authors: Juliano
authors: Laboratório de Piscicultura Marinha, Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco, Av. Dom Manoel de Medeiros, s/n, Dois Irmãos, 52171-900, Recife, PE, Brazil.
authors: Ronaldo Olivera
authors: RO
authors: Cavalli
authors: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade Universitária, 50670-420, Recife, PE, Brazil.
authors: Luiz Bezerra de
authors: LB
authors: Carvalho
authors: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade Universitária, 50670-420, Recife, PE, Brazil. Electronic address: ransoube@uol.com.br.
authors: Ranilson Souza
authors: RS
authors: Bezerra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002432:Cecum / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010473:Perciformes / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry*; D016415:Sequence Alignment; D013696:Temperature; D014866:Waste Products
chemical_list: D055767:Immobilized Proteins; D007477:Ions; D008670:Metals; D011480:Protease Inhibitors; D014866:Waste Products; D012685:Sepharose; D007611:Aprotinin; D003546:Cysteine Endopeptidases; C065940:alkaline peptidase
keywords: Digestive peptidase; Fish; Pyloric caecum; Rachycentron canadum; Trypsin