title: A chymotrypsin from the Digestive Tract of California Spiny Lobster, Panulirus interruptus: Purification and Biochemical Characterization.
abstract: A chymotrypsin was purified from the gastric juice of California spiny lobster (Panulirus interrutpus), using preparative electrophoresis and affinity chromatography on agarose-p-aminobenzamidine. The molecular mass was estimated by polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions to be 28 kDa. Chymotrypsin activity was totally inhibited by phenylmethylsulfonyl fluoride (PMSF) and chymostatin. Lobster chymotrypsin had optimal pH 7.0-8.0 and temperature of 55 °C. The enzyme is highly stable under a wide range of pH (retaining up to 80 % of activity after 1 h of incubation at pH 3.0, 5.0, and 12.0), showing higher stability at pH 8.0, and was inactivated after 20 min at 55 °C. Lobster chymotrypsin was able to hydrolyze protein substrates at as low as pH 3.0. These results are consistent with the findings of enzyme stability. Activity was assessed after incubation of enzyme with different organic solvents (in the range of 10-50 %); when tested in the presence of acetone, ethanol, propanol, and butanol, lobster chymotrypsin residual activity was >80 %; whereas in the presence of dimethyl sulfoxide (DMSO) and toluene, lobster chymotrypsin residual activity was <80 %. Deduced amino acid sequence, corroborated by mass spectrometry, was determined.
authors: Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Calle IPN 195, La Paz, B.C.S. 23096, Mexico.
authors: Betsaida
authors: B
authors: Bibo-Verdugo
authors: Liliana
authors: L
authors: Rojo-Arreola
authors: Maria A
authors: MA
authors: Navarrete-del-Toro
authors: Fernando
authors: F
authors: García-Carreño
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000032:analysis* / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004121:Dimethyl Sulfoxide; D004591:Electrophoresis, Polyacrylamide Gel; D005750:Gastric Juice / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides; D033521:Palinuridae / Q000737:chemistry*; D010664:Phenylmethylsulfonyl Fluoride; D059748:Proteolysis; D017422:Sequence Analysis, DNA; D013696:Temperature; D014050:Toluene
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D009842:Oligopeptides; D014050:Toluene; D010664:Phenylmethylsulfonyl Fluoride; C002101:chymostatin; D002918:Chymotrypsin; D004121:Dimethyl Sulfoxide