| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1187122 | Food Chemistry | 2013 | 6 Pages |
•Trypsin was isolated and purified from viscera of Pterygoplichthys disjunctivus.•It showed high activity at pH 5.5–11.0, 25–40 °C and 30% salt concentration.•Viscera from P. disjunctivus may be an important source of trypsin.•A biotechnological tool at low processing temperatures or high salt concentration.
Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe2+ > Hg2+ > Mn2+ > K+ > Mg2+ > Li+ > Cu2+). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). Km and kcat values were 0.13 mM and 1.46 s−1, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.
