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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-10, Vol.1033-1034, p.210-217
• Main Authors: França, Renata Cristina da Penha, Assis, Caio Rodrigo Dias, Santos, Juliana Ferreira, Torquato, Ricardo José Soares, Tanaka, Aparecida Sadae, Hirata, Izaura Yoshico, Assis, Diego Magno, Juliano, Maria Aparecida, Cavalli, Ronaldo Olivera, Carvalho Jr, Luiz Bezerra de, Bezerra, Ranilson Souza
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aprotinin - chemistry; Aprotinin - isolation & purification; Aprotinin - metabolism; Cattle; Cecum - enzymology; Cysteine Endopeptidases - isolation & purification; Digestive peptidase; Enzyme Stability - drug effects; Fish; Hydrogen-Ion Concentration; Immobilized Proteins - metabolism; Ions; Kinetics; Metals - pharmacology; Perciformes - metabolism; Protease Inhibitors - pharmacology; Pyloric caecum; Rachycentron canadum; Sepharose - chemistry; Sequence Alignment; Temperature; Trypsin; Waste Products
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.08.028

•BPTI immobilized onto sepharose as a purification tool for fish trypsins to overcome their production bottlenecks.•BPTI column allowed at least 15 reuse without loss of efficacy.•Activation energy and rate enhancement found for R. canadum purified enzyme can promote higher productivity. First data about catalytic proficiency of trypsin is provided.•The purified enzyme presents high analogy with psychrophilic peptidases without loss of thermal stability.•R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. 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−1mM−1. The catalytic proficiency of the purified enzyme was 2.75×1012M−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 109 to 1010-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.