کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1211863 | 1494024 | 2016 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
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چکیده انگلیسی
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 °C and 8.5, respectively. The enzyme was thermostable until 55 °C and its activity was strongly inhibited by the classic trypsin inhibitors N-Ï-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 Ï-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38 mM, kcat value was 3.14 sâ1, and kcat/km was 8.26 sâ1 mMâ1. The catalytic proficiency of the purified enzyme was 2.75 Ã 1012 Mâ1 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 molâ1 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Chromatography B - Volumes 1033â1034, 15 October 2016, Pages 210-217
Journal: Journal of Chromatography B - Volumes 1033â1034, 15 October 2016, Pages 210-217
نویسندگان
Renata Cristina da Penha França, Caio Rodrigo Dias Assis, Juliana Ferreira Santos, Ricardo José Soares Torquato, Aparecida Sadae Tanaka, Izaura Yoshico Hirata, Diego Magno Assis, Maria Aparecida Juliano, Ronaldo Olivera Cavalli,