Rapid purification of Streptomyces griseus trypsin by immobilized rice bran trypsin inhibitor.

Abstract

Pronase, a commercial. preparation of Streptomyces griseus protease, contains several proteinases and peptidases.^Among them, only one proteinase resembles bovine trypsin in its substrate specificity, response to inhibitors and physico-chemical properties.2~5) The purification of this enzyme, S.G. trypsin, has been performed in several ways,2'5~12) among which various affinity chromatography techniques, simple and rapid purification methods, were included. However, the purity of S.G. trypsin obtained by these simple methods was not quite satisfactory. We ourselves, however, have purified a trypsin inhibitor from rice bran, and have reported that the rice bran trypsin inhibitor (RBTI) inhibits only trypsin among serine-proteases.13) Therefore, it is very likely that immobilized RBTIselectively binds to S.G. trypsin in Pronase on affinity chromatography. Enzymeactivity toward BAEEor ATEEas substrates was determined by the procedure described by Narahashi1* with a slight modification, in a pH-stat (RTS 622, Radiometer) at 25°C under nitrogen gas stream. The reaction mixture contains 0.1 m KC1, 0.02m CaCl2 and 10mMBAEEor ATEE adjusted to pH 8.0. One unit of esterase activity is defined as the amount of enzyme which hydrolyzes 1 /miol substrate per minute under the conditions described above. BAPAhydrolytic activity was measured according to the method by Erlanger et al.1A) with a slight modification. The reaction was carried out at 37°C with a substrate concentration of 0.5mMin 0.1 m Tris-HCl buffer, pH 8.0, containing 0.01m CaCl2.Exopeptidase activities were measured with the substrates of 10mM Z-Gly-Leu, 2mMBz-Gly-Arg and 2.5mM Leu-Gly by the ninhydrin method of Yemmand Cocking.15) Concentration of S.G. trypsin in the solution was calculated from its absorption coefficient, E\0/°m = \l.?> (at 280nm), for pure enzyme.5) Thirty mg of RBTI prepared in our laboratory was immobilized by a conventional procedure16) using CNBractivated Sepharose 4B (3 g of dry gel, Pharmacia) and the immobilized RBTI was packed into a column (0.8 x 18 cm). The column was pre-equilibrated with 0.05 m Tris-HCl buffer, pH 8.0, containing 0.2m KC1 and 0.02m CaCl2. One hundred mg (dry material) of Pronase-P (Kaken Chemical Co.) dissolved in the above buffer was immediately applied to the column. The column was washed with the same buffer and then developed with pH gradient from 0.05 m Tris-HCl buffer, pH 8.0, containing 0.2m KCl and 0.02m CaCl2 (60ml) to 0.05m Na-acetate-HC1 buffer, pH 3.0, containing 0.2 m KCl and 0.02 m CaCl2 (60ml), followed by washing with 0.01 m HC1 containing 0.02m CaCl2. The elution profile is shown in Fig. 1. At pH 8.0, some proteins in Pronase were firmly adsorbed to the Fraction Isfumber Fig. 1. Affinity Chromatography of Pronase-P on a RBTI-Sepharose Column (0.8 x 18 cm). Elution buffers were successively changed. 1. 0.05m Tris-HCl, pH 8.0, containing 0.2m KC1 and 0.02m CaCl2 (100ml). 2. pH gradient; 0.05m Tris-HCl, pH 8.0, containing 0.2 m KG1 and 0.02 m CaCl2 (60ml)-0.05 m Na-acetate-HC1, pH 3.0, containing 0.2m K...