Separation, Purification And Characterization Of Kunitz Trypsin Inhibitor And Bowman-birk Trypsin Inhibitor From Soybean

Soybean trypsin inhibitor (STI) is one of the major anti-nutritional factors in soybeanwhich can affect protein utilization and digestion and cause many adverse physiologicalreactions. It is reported that there are more than five kinds of trypsin inhibitors in soybean, butonly separation methods of Kunitz trypsin inhibitor (KTI) and Bowman-Birk trypsin inhibitor(BBI) were reported at present. In terms of the experimental operation and effect we can observe,the domestic and overseas methods that had been reported had complicated separation processand low purification factor. Thus, in the current study, soybean seeds were used as raw materialsto explore both efficient and convenient purification methods of KTI and BBI so that we can laya theoretical foundation for further in-depth study on KTI and BBI.Soybean seeds were used as the raw materials. By the crude extract preparation of soybeanKunitz trypsin inhibitor and chromatographic methods, a higher efficiency separation andpurification method of KTI was studied and compared with the one-step method. Soybean seedsfrom Jiyu52were used as the raw materials. KTI was purified by multistep method, that is,crude extract of the Kunitz trypsin inhibitor was obtained by grinding, defatting, extraction ofphosphoric acid buffer, thermal denaturation and35%-75%ammonium sulfate precipitation,then a series of chromatographic methods including ion-exchange chromatography, affinitychromatography and Sephadex filtration were carried out; and one-step method, that is, one-steptrypsin affinity chromatography purification. The purified KTI was measured by SDS-PAGEelectrophoresis, MALDI-TOF analysis and N-terminal amino acid sequence analysis. Theresults showed that, from10g soybean seeds, through multistep method, purification fold of72.39times of KTI was obtained while the purification fold was only28.85times throughone-step purification. A series study on KTI showed that the inhibitor has a relative molecularweight of19.4kD by SDS-PAGE. The purified KTI obtained by multistep method was used forfurther purity analysis. The accurate molecular mass of this inhibitor was22907.51Da bydetermination of MALDI-TOF; Partial amino acid sequence of the purified protein from Edmandegration showed a high homology with various members of the Kunitz-inhibitor family. Aftercomparison and analysis of these results, we found purification factor of KTI from multistepmethod was much higher than that of one-step method, which suggested that multistep methodwas superior to one-step purification and the purified KTI from multistep method had a highhomology with numerous members of the the family of Kunitz-type inhibitors. A Bowman-Birk trypsin inhibitor from soybean seeds was isolated by a combination of50%alcohol precipitation, thermal treatment at65℃, isoelectric precipitation followed byacetone precipitation. Then this extract was purified on DE-52ion exchange and affinitychromatography. The purified BBI was measured by SDS-PAGE electrophoresis, MALDI-TOFanalysis and N-terminal amino acid sequence analysis. The results showed that the specificactivity of Bowman-Birk trypsin inhibitor was822.31U mg-1and purification fold of50.07wasobtained with a yield of69.34%. The purified BBI showed a relative molecular weight of9.8kDin SDS-PAGE electrophoresis. The accurate molecular mass of this inhibitor was8837.46Da bydetermination of MALDI-TOF. N-terminal sequence showed BBI had highly homologousamino acid sequences compared with other serine proteinase inhibitors, for example, homologyof BBI was80%,80%,73%,72%, and60%compared with Glycine max, Glycine soja, Vignamarina, Phaseolus grayanus and mung bean, respectively, which indicated that the purifiedmethod of BBI is ideal.