Physiological Changes of Rhodotorula glutinis Induced by an Acid Protease Inhibitor (S–PI)

An acid protease inhibitor (S–PI) accelerated the growth of Rhodotorula glutinis. This acceleration of the growth was observed only when the pH of the culture fluid lowered during the cultivation, but was not observed when the pH of the culture fluid was adjusted to 2.8 with NaOH. The increase in number of cells was closely related to cell deaths. The cell deaths were observed in an active growing state but not in a resting state. S–PI increased the DNA, RNA and protein content in the cell and decreased the lipid, especially the phospholipid content. When the yeast was cultured in an acidic medium having an initial pH of 1.8 in the presence of S–PI, the decrease of the phospholipid in the cell preceded, followed by the leakage of potassium ion, UV absorbing materials and free pool amino acid from the cell which were accompanied with the cell death. On the basis of these results, physiological changes occuring in the yeast cell are discussed.     

Complex Formation of Microbial Alkaline Protease Inhibitor (S-SI) with Subtilisin BPN′ and Its Properties

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′.     

Isolation and Structure-activity Relationship of Some Amylostatins (F-1b Fraction) Produced by Streptomyces diastaticus subsp. amylostaticus

In order to investigate the relationship between taste-blindness and its chemical structure, sensory tests on thioureas, guanidines, imidazoles and thiazoles, which are widely used as vulcanizing agents for rubber, were carried out. It was found that new compounds, which had an intermediate characteristic between bitter and taste-blind substances, were present in benzimidazole and benzothiazole compounds. When it is considered that these compounds have structural similarities and only slight structural changes were present in them, these results provide an indication of the bitter exhibition mechanism on a taste receptor.     

Affinity Chromatography of Acid Proteases on SPI Coupled to PAB-Cellulose

Tastes of Leu-Lys-Tyr (LKY) analogues, a series of potent angiotensin I converting enzyme (ACE) inhibitory peptides were evaluated. Some of these analogues were found to be sweet, such as Val–Lys-Tyr and Ala-Orn-Tyr. Furthermore, the structural requirements for sweetness or decreasing the bitterness were investigated by considerations of the structure–taste relationship with LKY analogues.     

Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

New Enzymatic Colorimetric Reactions of Benzoic Acid Derivatives with ABTS [2,2′-Azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] in the Presence of Laccase

New enzymatic colorimetric reactions of benzoic acid derivatives with 2,2′-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence of laccase are described. These laccase-catalyzed colorimetric reactions exhibited the characteristic colors arising from a strong absorption maximum in the visible region. Using these colorimetric reactions, benzoic acids e.g., 4-hydroxy-, 3-hydroxy-, and 3,5-diamino-benzoic acids, could be determined in micromolar amounts. These methods will offer broad applicability to those enzymatic assays which are designed to release benzoic acids.     

New Acid Proteases from Scytalidium lignicolum M-133

Rhamnan sulfate was extracted with boiling water from the cell wall of Monostroma nitidum, and the resulting extract was purified by ion-exchange and size-exclusion chromatography. The polysaccharide, which is regarded as a homopolysaccharide, was 6-fold more antithrombin-active than the heparin standard. The antithrombin activity was decreased, by desulfation, although the non-active product still retained 8% of the sulfate ester. A comparative structural study of the intact rhamnan sulfate and rhamnan, its desulfated product, by periodate oxidation, Smith degradation and methylation analysis revealed the rhamnan sulfate to consist of α-1,3-linked L-rhamnose residues, some of which were substituted with sulfate groups mainly at position O-2. Minor amounts of internal 1,2-linked rhamnose and branched rhamnose linkages were also detected.     

β-Glucosidase of Aspergillus aculeatus

Two kinds of cytochrome c oxidase were partially purified from iron-grown T. ferrooxidans. The first type (cytochrome c oxidase I) was easily solubilized without a detergent and had a pH-optimum at 3.0. The other (cytochrome c oxidase II) which was bound tightly to the cell membrane and solubilized with sodium dodecyl sulfate had a pH-optimum at 5.2. Each type was heat-sensitive and inhibited by cyanide and azide. Since the pH level of the bacterial iron oxidizing activity corresponded closely with the pH of cytochrome c oxidase I but not cytochrome c oxidase II, it was proposed that the former may play an important role in the iron oxidizing system.     

Binding of Pepsin Inhibitor (S-PI) and Pepsin

Relation between cellular phospholipids and L-glutamic acid excretion was investigated using Corynebacterium alkanolyticum GL–21 (a glycerol auxotroph).

When strain GL–21 was cultured in glycerol-limited medium which contained n-hexadecane, acetic acid or fructose as carbon source, there occurred the limitation of cellular phospholipid content and the over-accumulation of L-glutamic acid in the broth. Two-dimensional thin-layer chromatograms provided evidence that both the parent and the mutant strains contained the same phospholipids such as cardiolipin, phosphatidylethanolamine, phosphadityl-glycerol, phosphatidylinositol and phosphatidic acid. Limited supply of glycerol to the mutant did not greatly alter the proportions of the individual phospholipids.