Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste

Author da Penha Franca, Renata Cristina Google Scholar
Dias Assis, Caio Rodrigo Google Scholar
Santos, Juliana Ferreira Google Scholar
Torquato, Ricardo José Soares Autor UNIFESP Google Scholar
Tanaka, Aparecida Sadae Autor UNIFESP Google Scholar
Hirata, Izaura Yoshico Autor UNIFESP Google Scholar
Assis, Diego Magno Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Cavalli, Ronaldo Olivera Google Scholar
de Carvalho, Luiz Bezerra, Jr. Google Scholar
Bezerra, Ranilson Souza Google Scholar
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 24 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50 degrees C and 8.5, respectively. The enzyme was thermostable until 55 degrees C and its activity was strongly inhibited by the classic trypsin inhibitors N-rho-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 N-alpha-benzoyl-DL-arginine rho-nitroanilide hydrochloride (BApNA) as substrate, the apparent k(m) value of the purified trypsin was 0.38 mM, k(cat) value was 3.14s(-1), and k(cat)/k(m) was 8.26s(-1) mM(-1). The catalytic proficiency of the purified enzyme was 2.75 x 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 11.93 kcal mol(-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. (C) 2016 Elsevier B.V. All rights reserved.
Keywords Rachycentron canadum
Digestive peptidase
Pyloric caecum
Fish
Trypsin
xmlui.dri2xhtml.METS-1.0.item-coverage Amsterdam
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de Pernambuco (FACEPE)
Ministério da Pesca e Aquicultura (MPA)
Financiadora de Estudos e Projetos (FINEP)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
PETROBRAS Ambiental
Grant number FACEPE: IBPG 1563-2.08/08
CNPq: 559.759/2009-6
Date 2016
Published in Journal Of Chromatography B-Analytical Technologies In The Biomedical And Life Sciences. Amsterdam, v. 1033, p. 210-217, 2016.
ISSN 1570-0232 (Sherpa/Romeo, impact factor)
Publisher Elsevier Science Bv
Extent 210-217
Origin http://dx.doi.org/10.1016/j.jchromb.2016.08.028
Access rights Closed access
Type Article
Web of Science ID WOS:000385322600026
URI https://repositorio.unifesp.br/handle/11600/56895

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