Studies On Development, Characterization, Distribution And Zymogram Of Digestive Enzymes In Paddlefish Polyodon Spathula

A series of biochemical determination and electrophoretic observations have been conducted to analyze development, characterization, distribution and zymogram of protease,α-amylase and lipase in paddlefish Polyodon spathula. The research included study on development of digestive enzymes in paddlefish from 0 to 53 day after hatching, comparing activrty and characterization of protease, amylase and lipase in paddlefish with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus♀×Acipenser schrenki Brandt♂), a comparative study of a comparative study of enzymatic activity, distribution and characterization between pond-reared (with commercial feed) and cage-reared (with natural live food) paddlefish, effects of diet protein level on digestive enzyme activity of paddlefish and effects on tempareture and pH value on protease and amylase on paddlefish. The important results were in the followings:1. The alkaline protease activity was detected at 3 DAH and acid protease activity was quantifiable from eggs. Alkaline protease activity was consistently higher than acid protease activity.α-amylases activity was relatively stable during incubation period in workshop (12DAH～40DAH). High lipase activity indicated the importance of lipid utilization. Phosphatases activity significantly increased from 20DAH and alkaline phosphatases activity was higher than acid phosphatases activity. It is suggested that protease,α-amylase and phosphatase activities were increased with the ontogenesis of paddlefish and alkaline protease was important to paddlefish larval. Acid protease,α-amylase, acid and alkaline phosphatase activities were increased with growth decreasing and decreased with growth increasing when fish transferred to pond.2. Higher proteolytic activity was observed in the pH range 2.5–3.0 and at a pH of 7.0 for paddlefish; proteolytic activity of bighead carp had three peak in pH 4.0～4.5, pH 8.0～8.5, pH 11.0 and hybrid sturgeon indicated higher proteolytic activity in pH2.5～3.0 and pH 8.0～8.5. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp; while alkaline protease and amylase activity of paddlefish was significantly lower than those of bighead carp and hubrid sturgeon. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone (TPCK) was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone (TLCK). The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) analysis further showed that there were certain types of enzymes, especiallyα-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon.3. There was no significant difference observed for acid protease activity between pond and cage groups, whereas the activity of alkaline protease,α-amylase and lipase in the pond group were significantly lower than those in the cage group. Acid protease activity was detected in the esophagus, stomach and intestine, and alkaline protease demonstrated detectable activity in the duodenum and intestine; Theα-amylase showed activity across the entire digestive tract, whereas lipase activity was only detected in the stomach. The activity of acid protease in the intestine of the pond group was significantly higher than that in the cage group; however, there was no difference in enzyme activity of esophagus and stomach between both groups. The alkaline protease activity of pond group was significantly lower than that of cage group in both duodenum and intestine . In addition, theα-amylase activity of pond group was significantly lower than that of cage in the esophageal and duodenal sections; however, there was no difference in the activity in the stomach and intestine. Lipase demonstrated higher activity in the stomach of the pond group than that of the cage group.4,Both acid protease activity and alkaline protease activity of hepatopancreas were significantly increased with the increase of diet protein level, while alkaline protease activity of intestine was decreased with increase of diet protein level. The activity ofα-amylases in digestive tract was decreased with increase of diet protein level, whileα-amylases activity in hepatopancreas showed an opposite trend. There was no significant difference on lipase activity in all tested tissues among dietary protein treatments. The results indicated that there were significant effects of diet protein level on activities of acid protease, alkaline protease andα-amylase. The test indicated that 39.10% protein content suited the protein demands of paddlefish juvenile.5,The optimum temperature for acid protease and alkaline protease were 35～40℃and 35℃, respectively. Effect of temperature on amylase was not significant and there was no optimum temperature for amylase. The optimum pH value for acid protease, alkaline protease and amylase were2～3, >7.0, 5.5 and 8.0, respectively.