The Biological Properties Of Digestive Proteinases From Grass Carp (Ctenopharyngodon Idellus) Intestines

Enzymes, unsaturated fat acids, and antifreeze protein (AFP) are important functional compositions for the aquatic organisms adapting to cold environment. Enzymes, which are typical environment temperature dependent, play an important role in fish and other aquatic organisms. The digestive enzymes from fish or other aquatic organisms residing in the cold temperature environment have the properties of cold-adapted enzyme: performing high catalysis activity at low temperature; thermal unstable, denaturing fast at medium temperature; low Arrhenius active energy. In contrast, the digestive enzymes from tropic fish are thermal stable. Most of the investigated proteinases from fish and shrimp exhibit some properties of cold-adapted enzyme, but their adaptations to low temperature are discrete comparing to those occurring "real" cold enzymes.This thesis investigated the biological properties of proteinases from the intestines of grass carp, which is one of major fresh water fishes in China. The objectives of this project were evaluating the utilization potentiality of proteinases from intestines of grass carp, and intensively studying the digest physiology and nutrition of grass carp.Activity distributions of the main digestive enzymes of grass carp and black carp were studied. In the all digestive organs of grass carp and black carp, the acidic and basic protease, amylase and lipase were detected and were diversely distributed in the digestive organs of the two species. These enzymes are far more active in fish liver and pancreas than in intestine, and most of them distribute along the whole intestine of the two species. All the enzymes except amylase are more active in the forepart and middle part than the end of the intestines of the fishes (especially for the acidic protease of the black carp), but all the digestive enzymes are rather active in the distal part, especially for grass carp. The enzymes' activity distribution in intestines of the black carp are more sophisticated, and each has its own distribution patterns, and the enzymes distribute more uniformly in intestine of grass carp than that of black carp. The alkaline proteases activity is slightly higher in the forepart and middle parts than in the end of the Grass carp intestines. High protease activity in the whole intestines of grass carp may be one of the reasons why they can grow fast.The crude acidic and alkaline proteinases from the digestive tracts of grass carp were extracted, and some their properties have been studied. The thermostability assay results of the enzymes showed that the acidic proteinase lost 80% of its activity after heating at 50℃for 30 min, and the alkaline proteinase lost 80% of its activity after heating at 60℃for 30 min. The thermostability of alkaline proteinase showed the trend of environmental temperature adaptability.SDS-substrate-PAGE method was slightly modified and used to characterize classes and types of the grass carp alkaline proteinase. There were trypsins and metalloprotease (or elastase) in the grass carp intestine but not chymotrypsin. The simple zymogram might be more efficient especially to strong adaptability to the environments in that it resided and to its special food habit.Two types of trypsin isozymes were purified by gel filtration chromatography and ion-exchange chromatography. Their relative molecular masses were determined to be 26,400 and 30,750, respectively, using SDS-PAGE electrophoresis. The HPLC results showed that the enzymes were pure. All two isozymes could hydrolyze casein and BAEE, and be strongly inhibited by SBTI, PMSF and TLCK, but not by EDTA. They could be defined as trypsin according to their relative molecular mass, hydrolyzing characteristic and inhibition properties.The properties of the both purified trypsin isozymes (GT-A and GT-B) were determined. The optimum pH and temperature for GT-A are 8.0 and 40℃, and the optimum pH and temperature for GT-B are 8.5 and 45℃. The thermostability of GT-B is slightly higher than that of GT-A. Both trypsins in neutral phosphate buffer solution quickly lose their activities as temperature exceeds 65℃. The higher thermostability of both trypsins than that of fish trypsins from freezing conditions is an indication of adaptability to living environments' temperature for grass carp. Trypsins from Grass carp have high physiological efficiency of V_max/K_m and low Arrhenius activation energy (E_a). In the presence of CaCl₂, the activities of enzyme might not be influenced, however, their thermostability can be improved. Differencial scan calorimetry (DSC) analysis results showed that their heat-induced denature temperatures were 66.3℃(GT-A) and 67.3℃(GT-B), respectively. In the presence of Ca²⁺, the thermo denatured temperature is elevated by 6.7℃for GT-A and 7.1℃for GT-B. Thermal stabilities of both GT-A and GT-B were intermediate between Arctic and tropical fish species, and consistent with the wide range of water temperatures to which grass carp are exposed in most provinces of China.The amino acid composition and circular dichroism (CD) of two fish trypsin isozymes were determined. They contained low content of aromatic amino acids, cysteine and methionine, high content of serine and glycine. The content of acid amino acids was very high while that of alkaline amino acids was relatively low. Weak absorbance was read at wavelength of 280 nm for enzyme solution, but very high absorbance was read near 210 nm. And in the present of denaturant, the absorption increased significantly. According to "di-state model", the denaturation free energy (ΔG) was calculated to be 39.825 kJ/mol for GT-B. The results of CD indicated that both GT-A and GT-B contained small ratio ofα-helix,β-sheet, andβ-return but mostly random. The information of compositions and structures from both GT-A and GT-B can explain their physic-chemic and dynamical properties. Furthermore, it is verified that the active central of both GT-A and GT-B contains serine and histidine resides.The active components of acid proteases from Grass carp intestines were analyzed according to the substrate specificity of enzymes using substrate-PAGE electrophoresis. The acidic proteases from Grass carp intestines might be composed Cathepsin B, L or H and an enzyme which can hydrolyze bovine hemoglobin but can't hydrolyze N-bezoyl-1-argininamide (BAA). The crude acidic proteases extracted from Grass carp intestine were partially purified, the main active component of the partially purified acidic protease was an enzyme which can hydrolyze bovine hemoglobin while can't hydrolyze BAA. The optimal temperature and pH of the partially purified protease were 37℃and pH 2.3, respectively. The protease had poor thermostability, almost completely losing its activity when it was heated at 60℃for 30 min. It was strongly inhibited by pepstatin A and inhibited by ethylenediaminetetraacetate (EDTA), but it was not inhibited by PMST and SBTI.