Jiro Sawada

Studies on the Protease Constitution of Aspergillus oryzae: Part I. Systematic Separation and Purification of Proteases

In order to elucidate the protease constitution of Aspergillus oryzae, systematic separation of proteases was elaborated by sequential chromatography on Amberlite CG–50, DEAE-Sephadex A–50 and CM-cellulose. As the results, three kinds of proteases, that is, acid-, neutral- and alkaline proteases were isolated and purified in crystalline form except neutral one. Purified neutral protease could not be crystallized, but was confirmed to be homogeneous by ultracentrifugal analysis. Besides these proteases, a new protease which was unknown up to the present in the constitution of Asp. oryzae proteases, was first isolated and designated as “semi-alkaline protease” according to its optimal pH.

Studies on the Amylases of Paecilomyces subglobosus :Part I. Mycological Properties of Paecilomyces subglobosus sp. nov. TPR-3810 and -3811

Two strains of molds, which had a very close similarity with each other, and the acid-stable α-amylase activity of which was very high, were isolated from the air. Studies on the morphology of these strains revealed that, although they were fairly close to Paecilomyces varioti, they had some peculiar characteristics, and that their existence had not been reported hitherto. Therefore the authors concluded that the strains must belong to a new species in the genus Paecilomyces, and named them Paecilomyces subglobosus sp. nov. TPR-3810 and -3811, respectively, after their characteristic shape of the conidiospores.

Studies on the Acid-protease of Paecilomyces varioti Bainier TPR-220: Part I. Crystallization of the Acid-protease of Paecilomyces varioti Bainier TPR-220

A fungus, Paecilomyces varioti Bainier TPR-220 was found to produce an acid-protease on wheat bran culture (Koji). The protease showed the highest activity at pHs between 2.5 and 3.0 on casein as substrate. The enzyme was stable at pHs from 2.5 to 6.0 at 40°C for forty minutes. By crystallization from 50% acetone solution the specific activity of the protease was increased five times as high as that of the original crude enzyme preparation.

Studies on the Acid-protease of Paecilomyces varioti BAINIER TPR-220: Part IV. Physical and Chemical Properties of the Crystalline Acid-protease

Some physical and chemical properties were investigated of the crystalline acid-protease from Paecilomyces varioti BAINIER TPR-220. The sedimentation coefficient of the enzyme was calculated to be 2.7 × 10‒13 at 15°C and the molecular weight to be 37,300 by Archibald’s method. The isoelectric point of the enzyme protein was determined to lie at pH 3.8. The enzyme protein was consisted of 340 amino acid residues including only one residue of cysteine but excluding cystine. With the feature of amino acid composition of the protease acidic amino acids dominated over the basic ones.

Studies on the Acid-protease of Paecilomyces varioti Bainier TPR-220:Part II. Some Enzymic Properties of the Crystalline Acid-protease

The crystalline acid-protease of Paecilomyces varioti Bainier TPR-220 is most active toward casein as substrate, at pH 3.0 and 60°C, and stable at pH 3.0 to 6.0 below 40°C. The enzyme decomposes protein molecules into smaller fragments than pepsin does and is inhibited by p-chloromercuri-benzoate, monoiodoacetate, sodium lauryl sulfate, iodine, potassium permanganate, N-bromosuccinimide, bacitracin, nitrofurylacrylamide, and Hg+ ion, but affected neither by metal ion except Hg+ ion, nor metal chelating agent, soy bean trypsin inhibitor, potato-protease inhibitior, cysteine, diiso-propylfluorophosphate, cyanogen bromide, and heparin. The presence of Ca++, Co++, Cu++, Mg++, Sr++, and Zn++ ions prevents heat inactivation of the enzyme.

Studies on the Acid-Protease of Paecilomyces varioti Bainier TPR-220:Part III. The Specificity of the Crystalline Acid-Protease on Synthetic Substrates

The acid-protease of Paecilomyces varioti Bainier TPR-220 can hydrolyze carbobenzoxy-l-glutamyl-l-tyrosine, which is the substrate for pepsin, the acid-protease of Aspegillus saitoi by Yoshida and of Asp. oryzae by Nunokawa, and the trypsinogenkinase by Nakanishi, and benzoyl-l-argininamide, which is the substrate for trypsin, papain, and above two acid-proteases, optimally at pH 3.5 and 5.5 at 45°C, respectively. The Michaelis constant and activation energy of the enzyme are 4.08 × 10-3m and 9.17 × 103 cal/mol for the former, and 6.58 × 10-3m and 6.86 × 103 cal/mol for the latter, respectively.Other substrates for papain, chymotrypsin, and various exopeptidases are hardly hydrolyzed by the enzyme.