Study on the Properties of Polyphosphatases from Beef Semitendinosus and the Enzymic Reaction Systems
|School||Nanjing Agricultural College|
|Course||Of Food Science|
|Keywords||Beef semitendinosus PPase TPPase Enzymic property Polyphosphate (31)~P NMR|
Phosphates have been used as safe food additives in meat food processing, and the effect of phosphate complex was better than single phosphate. But phosphate can not play its role because of the hydrolysis. This paper have studied on the properties of purified pyrophosphatase and tripolyphosphatase from beef semitendinosus, then the phosphate complex (TSPP:STPP:HMP=2:1:1) and single phosphate added to the purified pyrophosphatase and tripolyphosphatase, using 31P NMR to observe the change processing of solution systems. The results showed that as followed,I. The purification of pyrophosphatase and the study on the properties of pyrophosphataseThe pyrophosphatase was isolated by means of homogenation,0.25 M sucrose and 0.6M NaCl extractions,50-70% ammonium sulfate precipitation, and ion-exchange chromatography on DEAE-52 cellulose column. The SDS-PAGE showed the relative molecular mass was 72 KDa. With sodium pyrophosphate as the substrate of pyrophosphatase, the time of preliminary velocity was 0-25min; the optimum pH was 7.4 and optimum temperature was 40℃; when Mg2+ concentration from 0 to 10mM the activity sharply increased, low concentration of Ca2+ had inhibit effect, when the concentration up to 25mM the pyrophosphates nearly have no activity; there was inhibit effect of EDAT-Na2 and EDAT-Na4 while the concentration from 0.1 mM to 10mM, but had no complete inhibition compare to Ca2+Ⅱ. The purification of tripolyphosphatase and the study on the properties of tripolyphosphataseThe tripolyphosphatase was purified by means of salt solution extraction, ion strength adjustment,35%-50% ammonium sulfate precipitation and ion-exchange chromatography on DEAE-52 cellulose column. The SDS-PAGE showed the relative molecular mass was 225KDa which was the same as myosin heavy chain. With sodium tripolyphosphate as the substrate of tripolyphosphatase the time of preliminary velocity was 0-25min; the optimum pH was 5.8 and optimum temperature was 28℃; Mg2+ was the activator when the concentration lower than 10mM, the activity was maintained by Mg2+ while more than 10mM, Ca2+、EDTA-Na2 and EDTA-Na4 had inhibit effects; When the concentration lower than 15mM the inhibit effect of EDTA-Na2 was better than EDAT-Na4.III. Influence of purified pyrophosphatase and tripolyphosphatase on polyphosphateThe experiments investigated the change processing of solution systems (pyrophosphatase, tripolyphosphatase, single phosphate or phosphate complex) using 31P NMR. The result showed that pyrophosphatase and pyrophosphatase catalyze the reaction of TPP-PP-Pi; with the same activity of pyrophosphatase and tripolyphosphatase, the hydrolysis rate of STPP faster than TSPP; the addition of STPP and HMP relatively inhibit the reaction process of PP-Pi; the addition of TSPP and HMP relatively inhibit the reaction process of TPP-PP. So the reason of different effects between single phosphate and phosphate complex was phosphate complex relatively inhibit the reaction of TPP-PP and PP-Pi compare to single phosphate.