Alkaline chymotrypsin from striped seabream (Lithognathus mormyrus) viscera: purification and characterization.
An alkaline chymotrypsin from the intestine of striped seabream (Lithognathus mormyrus) was purified by precipitation with ammonium sulfate, Sephadex G-100 gel filtration, Mono Q-Sepharose anion-exchange chromatography, ultrafiltration, second Sephadex G-100 gel filtration, and a second Mono Q-Sepharose anion-exchange chromatography with a 80-fold increase in specific activity. The molecular weight of the purified alkaline chymotrypsin was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. The enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 10.0-11.0 using succinyl-L-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPNA) as a substrate. The relative activities at pH 7.0 and 12.0 were about 66% and 45.5%, respectively. Further, the enzyme was extremely stable over a broad pH range (6.0-12.0). The optimum temperature for enzyme activity was 50 degrees C, and the enzyme displayed higher enzyme activity at low temperatures when compared to other enzymes. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulfonyl-fluoride (PMSF), a serine protein inhibitor, and N-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), a chymotrypsin specific inhibitor. The N-terminal amino acid sequence of the first nine amino acids was IVNGEEAVP. The chymotrypsin kinetic constants, Km and kcat on SAAPNA as a substrate, were 30.7 microM and 14.35 s(-1), respectively, while the catalytic efficiency kcat/Km was 0.465 microM(-1) s(-1). The high activity at high alkaline pH and low temperatures make this protease a potential candidate for future use in detergent processing industries.
El Hadj Ali
Nedra
N
Laboratoire de Genie Enzymatique et de Microbiologie, Ecole Nationale d'Ingenieurs de Sfax, Route Soukra Km 3.5, B.P. 1173-3038 Sfax, Tunisia.
Hmidet
Noomen
N
Zouari-Fakhfakh
Nahed
N
Ben Khaled
Hayet
H
Nasri
Moncef
M
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D021541:Sea Bream; D017386:Sequence Homology, Amino Acid
D002918:Chymotrypsin