Purification,Characterization And Molecular Cloning Of Trypsins From Mandarin Fish(Siniperca Chuatsi)

Trypsin (EC 3.4.21.4) is a member of the large family of serine proteinases, which specifically hydrolyze proteins and peptides at the carboxyl side of arginine and lysine residues and play a critical role physiologically and in the digestion of foods. As an enzyme which has high acitivy at low temperatures, trypsins have also been used in the food processing industry. However, till now,very little information regard trypsins from fresh water fish has been reported. In order to study the physiological and digestive functions of trypsins from fresh water fish and provide the basic information for used in the usage for food processing, we studied the purification, characterization and molecular cloning of trypsins from the pyloric caeca of mandarin fish, an important fresh water fish in China.An anionic form(Trypsin A) and a cationic form(Trypsin B) of trypsins from the pyloric caeca of mandarin fish(Siniperca chuatsi) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation, chromatographies on DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose chromatography.Purified trypsins revealed single bands on native-PAGE and SDS-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively on SDS-PAGE both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 and 40°C, respectively, and shared the same optimal pH at 8.5 using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45°C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective to these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca2+ and Mg2+ but inactivated by Fe2+, Zn2+, Mn2+, Cu2+, Al3+, Ba2+, Co2+ to different degrees. Apparent Kms of trypsin A and B were 2.18 and 1.88μM and Kcats of the two enzymes were 81.6 and 111.3 S-1 for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A cross reacted with the two purified enzymes, suggesting their homogeneity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous to trypsins from other species of fish.In the molecular cloning work, according to the conservative sequence of trypsins from different fish species and the N-terminal amino acid sequences of purified Trypsin B, we designed primers for cloning of trypsins from the pyloric caeca of mandarin fish by 3' RACE, 5' RACE and RT-PCR method. Two isoforms of trypsinogen genes were cloned and designated as TRS-A and TRS-B, respectively. The GenBank accession number was EU688996 and EU688997, and the protein accession number was ACD70339 and ACD70340。The cDNA clones of TRS-A and TRS-B showed that the open reading frame of two trypsinogens were both 729 bp in length encoding the protein of 242 amino acid residues with a signal peptide of 13 amino acid residues and active peptide of 7 amino acid residues. The deduced molecular masses of these two trypsinogens are 24111.16 Da and 24243.16 Da,respectively. Three residues(His-60, Asp-104 and Ser-196) forming the typical catalytic site of serine proteinases for functional activity were well conserved in the polypeptide sequence. Sequence alignment showed that the deduced primary structure of TRS-A and TRS-B shared identity of 84.71%. The identity of amino acid sequence of mandarin fish trypsins are over 80% to the trypsins from other species of fish. The amino acid composition, cleavage sites, secondary structure and tertiary structure of TRS-A and TRS-B were analyzed and predicted by bio-information software.