The Research Of Aspartic Protease Inhibitors From Soy Protein Hydrolyzed By Proteases

In this paper, the production process of aspartic protease inhibitor from soy protein after enzymatic hydrolysis has been optimized.SPI(soy aspartic proteinase inhibitor), with a IC50 value of 25.67μg/mL to pepsin(3000U) , was obtained through a series of purification steps.Experimental results showed that the highest inhibitory activity of hydrolysates could be got from soy protein(85℃water bath 20min) after papain hydrolysis in a dose of 3000U/g. Preliminary exploration of the minimum rage of the isoelecrtic point of SPI has been investigated, and the isoelectric focusing results showed that SPI has a isoelectric point between 4.4 and 5.1, it was concordant to most of the reported aspartic protease inhibitors.The purification method of SPI has been established.Soy protein was pretreated by heat treatment at high temperature to remove the indissoluble substance, and then isolated by DEAE Sepharose Fast Flow ion-exchange chromatography,Superdex Peptide 10/300 GL gel chromatography and SOURCE 15RPC reversed-phase chromatography.The purification fold was 62 and the inhibition activity was 254.2 IU /mg. The molecular weight distribution of SPI showed that SPI is a small peptide with a molecular mass of about 167, and most likely, it contains two peptides or peptide analogs.The basic properties of SPI has been studied.Results showed that SPI could react with pepsin in a vert short time and get the highest inhibitory effect at 20min.SPI has well thermal stability in a temperature rage from 20℃to 90℃and well pH stability in a pH rage from 2 to 10, the largest loss of inhibitory activity in different pH conditions was short than 10%.The experiment of protease resistance showed than SPI could not be decomposited by pepsin and other protease.The inhibitory zymogram of SPI showed that SPI has the highest inhibitory activity against aspartic proteases(pepsin, rennet), and has no inhibitory activity against other protease(serine proteases, thiol proteases, neutral protease, alkaline protease).Therefore, SPI was a aspartic protease inhibitor.Kinetic analysis showed that SPI did not occupy the active site of pepsin, it belongs to non-competitive inhibitor.It could be seen from the double reciprocal Lineweaver-Bunk curve that the Km value of pepsin stayed unchanged and the Vm value decreased.CD spectroscopy results showed that SPI has great impact to the secondary structure of pepsin.Due to the small molecular weight of SPI, it could be speculated that SPI could react with pepsin on many site of the pepsin molecule, thus lead to the great structure change of pepsin and affact the integration of pepsin and substrate.And it was consistent with the results of kinetic analysis.All the results of SPI showed that SPI was consistent with the protease inhibitors which were potential in medicinal use: natural product, low molecular weight(<1000Da), well thermostability and pH stability, high tolerance to other proteases in common use.