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API-2c, one of the alkaline protease inhibitors produced by Streptomvces griseoincarnatus strain No. KTo-250, interacts with subtilisin BPN′. The API-2c-subtilisin BPN′ complex was isolated by gel filtration on Sephadex G-100. Its homogeneity was determined by disc electrophoresis. From the results of molecular weight determination of the complex (MW 86,000) and of inhibitory assay of the API-2c released from the complex, it was shown that the complex was composed of one mole (two subunits) of API-2c and two moles of subtilisin BPN′.The inhibitory activity was gradually released from the complex by dialysis against 0.05 n HC1 at 4°C. The inhibitor released showed a different mobility from virgin API-2c. These results suggest that API-2c was modified to API-2c* which peptide linkage in the reactive site was specifically cleaved.

API-2 degrading protease produced by Streptomyces griseoincarnatus strain No. KTo-250 was purified. Optimal pH of the protease was 9.0 which was the same as that of API-2b→ API-2c converting protease. But the protease was clearly distinguishable from API-2b→ API-2c converting protease: The molecular weight of the degrading protease and the converting protease were 28,000 and 48,000, respectively, and the former was inhibited by AP-I and S-SI, whereas the latter was not inhibited by either. Moreover, API-2 degrading protease degraded or digested API-2c, although API-2b→API-2c converting protease did not degrade it.

Further studies were made on muscle alkaline protease and, at the same time, its properties were compared with those of the protease in peritoneal cavity cells. Both enzymes showed similar properties. Their molecular weight was 22,000, optimum temperature 57°C, optimum pH 9.0~10.5. They were slightly inhibited by soybean trypsin inhibitor. Tosylphenylalaninechloromethylketone inhibited both enzymes but tosyllysinechloromethylketone did not. Both enzymes cleaved peptide bonds Tyr (16)-Leu(17), Phe(24)-Phe(25) of oxidized B chain of insulin.

The use of acetic acid as a carbon source in alkaline protease fermentation was examined. Acetic acid was a good carbon source and yielded a great deal of alkaline protease.Acetic acid has advantages over ordinary carbon sources such as starch and glucose in that it can be supplied to culturing liquid as much as needed to perform the fermentation efficiently, that it has a function to control the pH of culturing liquid at a constant level and that it was obtained at lower price.The maximum proteolytic activity attained was 1.6 × 104 units/ml (11.4 mg-enzyme/ml).

Microbial alkaline protease inhibitor, S–SI, was investigated on the interaction with subtilisin BPN′ Inhibitory equivalent of S–SI to subtilisin BPN′ was determined that one molecule of S–SI (MW = 23,000) inhibited two molecules of subtilisin BPN′ (MW = 27,700). The S–SI-subtilisin BPN′ complex was isolated by gel filtration on Sephadex G–100 and rhombic crystals were obtained. DIP- and ZAGPCK-subtilisin BPN′ did not form such complex with S–SI. Homogeneity of the complex was determined by disc electrophoresis. The isoelectric point of the complex was pH 5.5. Assay of S–SI dissociated and amino acid analysis of the complex indicated that one subunit (a half molecule) of S–SI was combined with one molecule of subtilisin BPN′ From molecular weight determination, it was clarified that the complex was composed of one molecule (consist of 2 subunits) of S–SI and two molecules of subtilisin BPN′.

To establish a novel process for the economical production of alkaline protease from n-paraffins by fungi, the variety of fungi were screened for the production of alkaline protease from n-paraffins and Fusarium sp. S–19–5 was selected as an excellent strain, and then the attempt was made to obtain some mutants capable of producing alkaline protease in high yield. A kabicidin resistant mutant No5-128B, was successfully obtained from Fusarium sp. S–19–5 by treatment with ultraviolet light. This mutant No5–128B showed high productivity of alkaline protease, about ten times that of the parent strain. The morphological characteristics and growth response to Kabicidin of the mutant No5–128B were compared with those of the parent stain.

Zymolyase was purified from Zymolyase-60,000 by ion-exchange chromatography with sugar and gel filtration. Zymolyase was separated into the two protein fractions A and B. Neither alone could lyse yeast cells, but together showed high lytic activity. Zymolyase A and B were β-1,3-glucanase and alkaline protease, respectively. On stepwise treatment of yeast cells with the enzymes, yeast cells were lysed only by treatment with Zymolyase A after pretreatment with Zymolyase B. Zymolyase A lysed yeast cells in the presence of 2-mercaptoethanol, but B could not even at high concentration. Zymolyase B decreased the turbidity of a yeast cell suspension by about 13%.

Among three alkaline protease inhibitors (API-2a, -2b, -2c) produced by Streptomyces griseoincarnatus strain No. KTo-250, API-2b was converted to API-2c in the growing system.The cultural conditions were examined exclusively for the production of API-2b→ API-2c converting protease in the culture filtrate. The protease was purified about 1080-folds by salting-out with ammonium sulfate, column chromatography on DEAE-cellulose and gel filtration on Sephadex G–100.The optimal and maximal caseinolytic activities of the protease were around pH 9.0 and at 28°C, respectively. The protease activity was inhibited by EDTA and DFP, but not by PCMB, o-phenanthroline, TPCK, TLCK, AP-I and S-SI. The protease was a DFP and EDTA-sensitive alkaline protease, and it required Ca2+ ion for its activity and stability.

The specificity of highly purified alkaline proteinase B (EC 3.4.21.14) from thermophilic Streptomyces rectus var. proteolyticus was investigated with an oxidized insulin B chain. Hydrolysis of the oxidized insulin B chain in a 4-hr incubation was observed mainly at three peptide bonds (Phe24-Phe25, Leu15-Tyr16 and Leu11-Val12) and additionally at six others (Leu6-CySO3H7, Gln4-His5, Leu17-Val18, His5-Leu6, Glu13-Ala14, Asn3-Gln4).Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr4-Ile5 bond. Hydrolysis of proangiotension (formerly designated angiotensin I) was observed at the Tyr4-Ile5 and Phe8-His9 bonds.

A novel process for the microbial production of alkaline protease on an industrial scale was successfully established by using a kabicidin resistant mutant, No. 5–128B, derived from Fusarium sp. S–19–5. The most suitable carbon source for producing alkaline protease was n-paraffins (C10~C14) and the effective nitrogen source was dried-yeast cells containing no nucleic acid, the optimum concentrations being 12.5% (w/v) and 7.0% (w/v), respectively. The optimal temperature and initial pH for protease production were 24°C and 6.0, respectively. Under the optimal conditions using a shaker flask mutant No. 5–128B produced 41000 PU/ml of alkaline protease, which corresponded to about 10 times the amount produced by the parent strain. The relation between the high ability to produce alkaline protease and the resistance to kabicidin, a polyene antibiotic, is discussed.

EI complex formation of AP–I, existing as dimer, with subtilisin BPN’ was investigated in detail.By changing the E/I2 ratio in the reaction mixture, two types of EI complex were recognized; E2I2 complex which was composed of two moles of subtilisin BPN’ and one mole (two subunits) of AP–I, and EI2 complex which was composed of one mole of the enzyme and one mole of AP–I. Their existence was demonstrated by gel filtration, inhibitory equivalent, disc electrophoresis and isoleucine content in the complexes.The former complex is thought to be a final and stable complex, and the latter to be an intermediate or transient type of complex.

The alkaline proteinase from Aspergillus sydowi was markedly protected from inactivation by the presence of Ca++ in the enzyme solution. The protective effect of Ca++ was influenced remarkably by the pH values of the enzyme solution, i.e., optimum concentrations of Ca++ for the protective effect at pH 7.1, 7.5 and 7.8 were 10-2, 10-3 and 10-4M, respectively. Conversely, at higher pH values such as 9.0, Ca++ accelerated the rate of inactivation. There was a parallelism between the loss in activity and the increase in ninhydrin-positive material in the enzyme solution. The proteinase acted on various denaturated proteins, but not on native proteins. In digestion of casein by the proteinase, 92% of nitrogen was turned into soluble form in 0.2M trichloroacetic acid solution, with 14_??_17% of peptide bonds being hydrolyzed. Casein hydrolyzed with the Asp. sydowi proteinase was further hydrolyzed by Pen. chryso-genum, B. subtilis or St. griseus proteinases, which further increased the free amino residues in the reaction mixtures. On the contrary, the Asp. sydowi proteinase reacted only slight-ly on casein hydrolyzed by the above-mentioned proteinases.

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be...

(1977). Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis. Agricultural and Biological Chemistry: Vol. 41, No. 7, pp. 1309-1310.