A laundry detergent-stable alkaline trypsin from striped seabream (Lithognathus mormyrus) viscera: purification and characterization.
An alkaline trypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified and characterized. The enzyme was purified to homogeneity by precipitation with ammonium sulfate, Sephadex G-100 gel filtration and CM-Sephadex cation-exchange chromatography, with a 24.9-fold increase in specific activity and 13% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The purified trypsin appeared as a single band on native PAGE. Interestingly, the enzyme was highly active over a wide range of pH from 8.0 to 11.0, with an optimum at pH 10.0 using Nalpha-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 8.0, 11.0, and 12.0 were 73%, 67% and 50.4%, respectively. The enzyme was extremely stable over a broad pH range (5.0-12.0). The optimum temperature for enzyme activity was 50 degrees C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI). In addition, the enzyme showed excellent stability toward various surfactants and bleache agents and compatibility with some commercial solid and liquid detergents. The trypsin kinetic constants, Km and kcat of the enzyme for BAPNA, were 0.29 mM and 1.36 s(-1), respectively, while the catalytic efficiency kcat/Km was 4.68 s(-1) mM(-1).
Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d'Ingenieurs de Sfax, B.P. W, 3038 Sfax, Tunisia.
Nedra
N
El Hadj Ali
Noomen
N
Hmidet
Ali
A
Bougatef
Rim
R
Nasri
Moncef
M
Nasri
D000818:Animals; D003902:Detergents / Q000494:pharmacology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D021541:Sea Bream; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014781:Viscera / Q000201:enzymology*
D003902:Detergents; D014361:Trypsin Inhibitors; D014357:Trypsin