Purification And Characterization Of Trypsin And Chymotrypsins From Hepatopancreas Of Crucian Carp (Carassius Auratus)

Trypsin (EC 3.4.21.4) and Chymotrypsin (EC 3.4.21.1) are endopeptidases and are two members of the large family of serine proteinases. Trypsin specifically hydrolyzes proteins and peptides at the carboxyl side of arginine and lysine residues. Chymotrypsin cleaves on the carboxyl side of phenylalanine, tyrosine and tryptophan residues. Trypsin and Chymotrypsin are not only play important physiological and digestive functions in fish body, but also play a critical role in protein digestion for food processing. As fish enzymes that have high acitivy at low temperatures, trypsin and chymotrypsin have also been used in food processing industry. However, till now,information about fish chymotrypsins and trypsins from fresh water fish are less available. In order to study the physiological and digestive functions of trypsins and chymotrypsins from fresh water fish and provide the basic information for application in food processing, we studied the purification, characterization and preparation of polyclonal antibodies against chymotrypsin B and trypsin from the hpatopancreas of crucian carp, an important fresh water fish in China.An anionic form (Chymotrypsin A) and a cationic form (Chymotrypsin B) of chymotrypsins and an anionic form trypsin from the hepatopancreas of crucian carp (Carassius auratus) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation, column chromatographies on DEAE-Sepharose, Sephacryl S-200 HR, Phenyl-Sepharose or SP-Sepharose.Purified chymotrypsins and trypsin revealed single bands on native-PAGE and SDS-PAGE. The molecular weights of chymotrypsin A, chymotrypsin B and trypsin were 28 kDa, 27 kDa and 21 kDa, respectively on SDS-PAGE both under reducing and non-reducing conditions. Chymotrypsin A and chymotrypsin B exhibited maximal activity at 40°C and 50°C, respectively, and optimal pHs were 7.5 and 8.0 using Suc-Leu-Leu-Val-Tyr-MCA as substrate. However, trypsin respectively revealed maximal activity at 35°C and pH 8.5, using Boc-Phe-Ser-Arg- MCA as substrate. The two chymotrypsins and trypsin were stable up to 40°C and in the pH range from 5.5 to 11.0. Serine proteinases inhibitors are effective to chymotrypsins and trypsin and their susceptibilities were similar. Chymotrypsin A, chymotrypsin B and trypsins were activated by metal ions such as Ca2+ and Mg2+ but inactivated by Fe2+, Zn2+, Mn2+, Cu2+ and Cd2+ to different degrees. Apparent Kms of chymotrypsin A and B were 1.4 and 0.5μmol/L and Kcats of the two enzymes were 2.7 and 3.4 S-1 using Suc-Leu-Leu-Val-Tyr-MCA as substrate, respectively. Apparent Km and Kcat of trypsin were 0.8μmol/L and 203.5 s-1, respectively using Boc-Phe-Ser-Arg-MCA as substrate. Immunoblotting analysis using anti-chymotrypsin B weakly cross reacted with chymotrypsin A, suggesting their homogeneity. Immunoblotting analysis using anti-common carp trypsin A cross reacted with the purified trypsin, suggesting common carp trypsin A is homogenous to crucian carp trypsin.