Effect of Spacer Length on Recovery of Acid Protease from Raw Shoyu by Pepstatin-Sepharose Affinity Chromatography

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In order to investigate the effect of the spacer in pepstatin-Sepharose on adsorption and elution of acid protease (AcP) in raw shoyu (unpasteurized soy sauce), a homologous pepstatin-aminoalkyl agarose series, (pepstatin-NH2(CH2)n-Sepharose), that varied as to the length of the hydrocarbon chains was synthesized. When raw shoyu containing many kinds of proteases was subjected to affinity chromatography on these pepstatin-Cn-Sepharoses (n=2, 4, 6, 8, 10 and 12), all of them adsorbed AcP. With increasing length of the spacer up to 6, more and more AcP became adsorbed onto the pepstatin-Cn-Sepharose, whereas with decreasing length of the spacer, more and more AcP was eluted with 0.05M acetate buffer (pH 3) containing 2M urea. The AcP was purified in one step from raw shoyu and did not have any carboxypeptidase activity. Some properties of the major component of the eluted AcPs were as follows: molecular weight, 6.7×104, on gel nitration with TSK-G3000SW, optimum pH for activation of trypsinogen, 3.5, optimum pH for hydrolysis of hemoglobin, 2.75, and the Ki value toward pepstatin, 1.0×10-8M.
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