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Purification and Characterization of Trypsin and Chymotrypsins from Hepatopancreas of Crucian Carp (Carassius Auratus)

Author: YangFeng
Tutor: SuWenJin；CaoMinJie
School: Jimei University
Course: Of Food Science
Keywords: Trypsin Chymotrypsin Carp Purification Western blot Homology Properties of
CLC: S917.4
Type: Master's thesis
Year: 2009
Downloads: 210
Quote: 3
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Abstract


Trypsin (Trypsin, EC 3.4.21.4) and chymotrypsin (Chymotrypsin, EC 3.4.21.1) is a serine protease family hydrolase, endopeptidase enzyme. Trypsin specific cleavage of the basic amino acid residues (Lys, Arg) the carboxyl side chain of a peptide bond; the chymotrypsin specificity of the peptide bond hydrolysis of the carboxyl side chains of Phe, Trp, and Tyr, and other hydrophobic amino acid residues. Trypsin and chymotrypsin not only plays an important physiological functions in fish, and have high activity and stability at low temperatures, potentially important application value tool enzyme. Currently, the more at home and abroad about the fish trypsin, but little research chymotrypsin. This topic freshwater carp for the first time as the research object, its liver pancreas in pancreatic chymotrypsin and trypsin were isolated and purified to study its enzymatic properties, and prepared-antichymotrypsin and trypsin polyclonal antibody, provides a theoretical foundation for the future study of freshwater fish chymotrypsin and trypsin physiological functions such enzymes in biological, pharmaceutical, and food processing. Subject to separation and purification of chymotrypsin and trypsin method comprising salting out of ammonium sulfate fractionation, DEAE-Sepharose anion exchange chromatography, Sephacryl S-200 gel filtration chromatography, SP-Sepharose cation exchange chromatography and Phenyl-Sepharose hydrophobic chromatography. This study has been two types of chymotrypsin the (anionic Chymotrypsin Chymotrypsin A and cationic chymotrypsin Chymotrypsin B) and an anionic trypsin protease. SDS-PAGE and Native-PAGE results showed that the Chymotrypsin A, Chymotrypsin B and Trypsin are highly purified. SDS-PAGE showed Chymotrypsin A, Chymotrypsin B and Trypsin molecular weight of 28 kDa, 27 kDa and 21 kDa. Suc-Leu-Leu-Val-Tyr-MCA as a substrate A Chymotrypsin Chymotrypsin B the optimum temperature were 40 ° C and 50 ° C, the optimum pH were pH 7.5 and pH 8.5; When the hydrolysis bottom when objects Boc-Phe-Ser-Arg-MCA, the the Trypsin optimum temperature and pH value of 35 ° C and pH 8.5. Chymotrypsin A, Chymotrypsin B and Trypsin at 40 ° C or less stable, the tolerance range of pH values ??of 5.5 to 11.0. Chymotrypsin A, Chymotrypsin B and Trypsin, a serine protease inhibitor produced a strong inhibition, and similar to the inhibitory effect of the two enzymes. Trypsin substrate specificity experiments showed that the strongest enzyme decomposing ability of the fluorescent substrate Boc-Gln-Arg-Arg-MCA. Chymotrypsin A, Chymotrypsin B and Trypsin Ca2 and Mg2 ions activated by Fe2, Zn2, Mn2, Cu2, Ba2, and Cd2 ion suppression to some extent. Kinetic experiments showed that the Suc-Leu-Leu-Val-Tyr-MCA as substrate Chymotrypsin A Chymotrypsin B The Km values ??were 1.4 and 0.5μmol / L, corresponding to the Kcat value were 2.7 and 3.4 s- 1; Km value and Kcat value of Trypsin to 0.8μmol / L, and 203.5 s-1, respectively. The immunoblotting experiments Chymotrypsin A Chymotrypsin B Chymotrypsin B antibody and anti-carp positive reaction; Trypsin positive reaction with anti-carp trypsin antibody.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquatic basic science > Aquatic Biology > Aquatic Zoology
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